Chapter I. Identification and Sequence analysis of largest Subunit of Origin Recognition Complex, PfORC1

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2 Chapter I Identification and Sequence analysis of largest Subunit of Origin Recognition Complex, PfORC1

3 Results: ChapterI 1.1 In siliico analysis of PfORC1 DNA replication is poorly understood in P. Jalciparum. In order to look for the presence of replication initiation proteins in Plasmodium, P. Jalciparum genomic database (PLASMODB.ORG) was searched thoroughly using S. cerevisiae ORC subunits as queries. Analysis of P. Jalciparum genomic database revealed the presence of few putative ORC like proteins namely pforc1, pforc2 and pforc5 (as shown in table 8). No other ORC subunits have been found in PlasmoDB yet. It is interesting to note that among ORC subunits ORC 1 is the largest subunit that is regulated through cell cycle (DePamphilis, 2005; Mendez et al., 2002). Although few putative ORC subunits have been identified in Plasmodium, we will focus mostly on the putative ORC1 homolog here to investigate its function in P. Jalciparum DNA replication initiation and control. Table 8: ORC like molecules in Plasmodiumfalciparnm Gene name Accession no. Database Homologous region PtDRCl PFL0150w PlasmoDB C-terminus PtDRC2 MAL7Pl.21 PlasmoDB C-terminus PtDRC5 PFB0720c PlasmoDB C-terminus The Plasmodium database (PlasmoDB) predicts an ORF corresponding to the largest subunit of ORC, pforc1 that corresponds to a protein of 1189 amino acids having a calculated relative molecular mass of -140 kda and an estimated theoretical pi of The ORF has no introns within it. pforc1 gene is highly AT-rich and contains low complexity regions in the entire protein. Alignment of amino acid sequences of ORC 1 proteins from different organisms was performed using Clustal W as shown in Figure 7 and the -74 -

4 Results: Chapter! positions of conserved sequence blocks and potential structural motifs within PfORC1 are illustrated schematically in Figure 8. The N-terminus of PfORC1 shows a long extension that does not share homology with its counterparts. The C-terminus of PfORC1 shows considerable homology with other counterparts. The primary structure of PfORC1 shows maximum homology with S. cerevisiae ORC1 (57%) followed by human ORC1 (54%) in the overlapping region at C-terminus. The highest degree of sequence similarity between PfORC 1 and other related proteins is around within a nucleotide-binding domain of Rossman fold family. ORC1 is a member of a large superfamily of ATPases known as ATPases associated with various cellular activities (AAA) proteins (Beyer, 1997). Other members of AAA+ subclass are Cdc6, ORC4, ORCS, replication factor C (RFC), bacterial DnaA, DnaB, the bacterial gamma and epsilon clamp-loader subunits etc. Homology among these proteins is restricted to the C-terminal portion of the proteins, which bears the Walker A and Walker B motifs. Although P. Jalciparum genome database failed to identify any gene homologus to cdc6, it is interesting to note that PfORC1 share a significant identity (36%) and similarity (55%) with HsCdc6 sequence at its C-terminus. PlasmoDB predicts PfORC 1 as putative Cdc6 counterpart in P. JaZciparum based on its homology with cdc6 gene sequences with different organisms. In order to compare the conserved C-terminus of pforcl with Cdc6 sequences of different organisms, we have examined the phylogenetic relationship between PfORC1 AAA+ domain and Cdc6 proteins of different organisms. To accomplish this goal, a phylogenetic tree was generated using sequences of Cdc6 from different organisms and was compared with PfORC1 C-terminus domain which possesses the homology with Cdc6 sequence. The PHYLIP tree illustrates that the Cdc6 sequence of Sulfolobus solfataricus is closely related with PfORC 1 conserved C-terminal domain. In S. solfataricus ORC1jCdc6 protein -75 -

5 Results: ChapterI plays the role of replication initiator as well as helps in the loading of MCM complex onto the replication origin (Duggin and Bell, 2006). pfor.c 1 is distantly related with Cdc6 sequence of Arabidopsis thaliana and S. cerevisiae (Figure 8). CLUSTALW analysis oforel from different organisms DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 MTPKKKIFQNFQANDNEILSPTKKGIKLNVSKLNILNFENTIITKEKTNYEYKASLNKEI 60 DmORC1 HsORC1 AtORC1 ScORC1 PfORC MVNKENARSVGQQVKWIGSQDELPPVKNLEHKNVYFYQKCIYGPLTLS MAHYPTRLKTRKTYSWVGRPLLDRKLHYQTYREMCVKTEGCSTEIHIQ MASSLSSKAKTFKSPTKTPTKMY MAKTLKDLQGWEIITTDEQGNIIDGGQKRLRRRGAKTEHYLKRSSDGIKLG 51 DEVLNNNNIINTHNNKKNNLNLYDYNNIKNSTHEFYIDLNEQNKQTIKYNDNKFTPINKK 120 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 VGDFILVSNADAAEPDTV SGCDVARILHMYELRELTDREPCRAIVQWYSWPKA 101 IGQFVLIEGDDDENP YVAKLLELFED-DSDPPPKKRARVQWFVRFCE 94 RKSYLSPSSTSLTPP QTPETLTPLRRSSRHVSRKIN 59 RGDSVVMHNEAAGTYS VYMIQELRLNTLNNVVELWALTYLR 92 EKYNLDETSSSSISSSLTNISSSLTNISSSLTNISSSLSNSLDEKKKKKKIKKNNSTIIN 180 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 IPHNKYDDDEVAIDFSLEVIEEH----RPYDNDVALGAIYRKCIVLEGTSKTSAEEILKR 157 VPACKRHLLGRKPGAQEIFWYDY----PACDSNINAETIIGLVRVIPLAPKDVVPTNLKN 150 LGNDPIDLPGKESVEEINLIRKP----RKRTNDIVVAEKSKKKKIDPEVSFSPVSPIRSE 115 WFEVNPLAHYRQFNPDANILNRP----LNYYNKLFSETANKNELYLTAELAELQLFNFIR 148 ILNNNNNNSNIHHNNKHNIYNKYNISKKPQNKEIHTLPSNHQIKKKSNNTYNTCQQKMKK 240 DrnORC1 HsORC1 AtORC1 ScORC1 PfORC1 HANKLKSTACPMFVSRYRFVKVKRSYRLIPLEIHLEQPEDNARPTRSSRKSLTAHRES EK TLFVKLSWNEKKFRPLSSELFAELN----KPQESAAKCQKPVRAK TK KTKKKKRVYYNKVEFDET------EFEIGDDVYVKRTE VAN VMDGSKWEVLKGNVDPERD FTVRYICEPTGEK NISKKNTHSIKNNQNDKNKEKNKEKDKNIKKDRDKDIQTKRTSHQSQDQNNHFERRILRS 300 DmORC1 HsORC1 AtORC1 ScORCl PfORC KRSISARHDDTAGNKGSSVEKRRRASMAASSSVEFIDVNSFICE SKSAESPSWTPAEHVAKRIESRHSASKSRQTPTHPLTP DANPDEEEEEDPEIEDCQICFKSHTNTIMIECDDCLGGFHLNCL FVDINIEDVKAYIKKVEPREAQEYLKDLTLPSKK YTRNNDNVKNNLKNNINNNNTLKRSSQSVRIDSDLSSAHQNKRIKYDEKNIIHRNNNNNN 360 DmORC1 HsORC1 NKVSPIKIVGGRSVVRLSEKKNAPEINANYLPASPLTEKNAKVETPKSRASAARRNLNLS 319 -RARKRLELGNLGNPQMSQQTSCASLDS---PGRIKRKVAFSEITSPSKRSQPDKLQTLS

6 Results: ChapterI AtORC1 ScORC1 PfORC1 DmORC1 HsORC1 AtORC1 ScORCl PfORC1 KPPLKEVPEGDWICQFCEVKKSGQTLVVVPKPPEGKKLARTMKEKLLSSDLWAARIEKLW 253 -KEIKRGPQKKDKATQTAQISDAETRATDITDNEDGNEDESSDYESPSDIDVSEDMDSGE 276 NNNNKTTSNNHNKNNKINNNNPSENYKKQTDTKHTNNTQNNKYNKTKTTNTFKHPSKDHT 420 LDRGADTTADSDCLNYSIVQQTPDPKTPSNDMKIKLRLSERRRSVRLASMDVDPLSLEEA 379 PALKAPEKTRETGLSYTEDDKKASP EHRIILRTRlAASKTIDIREE 333 KEVDDGVYWlRARWYMIPEETVLGR QRHNLKRELYLTNDFADIEME 299 ISADELEEEEDEEEDEDEEEKEARH TNSPRKRGRKIKLGKDDIDAS 322 DVNTKTFKSRYINTYTMEEVQKSIKSNTIKLVQENSCEYQDGIIYESIQINDEEYSIGED 480 DmORC1 HsORC1 AtORC1 ScORCl PfORC1 VQEPNAQGRKRLGVANGDIYHTPTK RTLTPISGGQRSSVVPSVILKPENI CVLR------HCFVKCPKEFSKASN VQPPP KKRGRKPKDPSK VLIFYTPNNNTYNAKSDDKKNQNNNNIKENIYLLKKGKISSFYKSTNSKVIEVEICFYYD 540 DmORC1 HsORC1 AtORC1 ScORCl PfORC1 KSKEPLESAAATEQTPSTRRKSI LKSATSRLAEGTPRRSIHLSNIVEQRVF 455 KKRDAKEAKAQNEATSTPHR IRRKSSVLTMNRIRQQLRFLGNSKS DGDDVFLCEYEYDVHWGSFK RVAELADGD PRQMLLISSCRANNTPVIRK ESDEQRlRELEKKQTSRRCKEDFNIYLDDDTKYYNLLGNIHFTILDANYIYKKIYVYNEI 600 DmORC1 HsORC1 AtORC1 ScORCl PfORC1 EDDEIISTPKRGR DQEEKEILPAAE EDSDQEWNGRKE FTKKNVARAKK ETFEEDTHARKGKNKFLCTHFIKDKEDRLCFIPNEDHWDNLVLGSSDLYYYFANEKKLNK 660 DmORC1 HsORC1 AtORCl ScORC1 PfORC SKKTVQDNDEDYSPKKSVQKTPTRTRRSSTTTKTATTPSKGITTATATPMTP ISDSSSDEEEASTPPLPRRAPRTVSRNLRSSLKSSLHTLTKVPKKSLKPRTP EEIDYSDEEIEFDDEESVRGVSKSKRGGANSRKGRFFGLEKVGMKRIPEHVR KYTPFSKRFKSlAAIPDLTSLPEFYGNSSELMASRFENKLKTTQKHQIVETI 422 NKSLKLIIEKLKINDKINDTQANQKKNNKKEYMNKAQTTTNVKANTHTKTLNDHNKSKTT 720 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 SQKMKKlRAGELSPSM QQRTDLPAKDSSKSELQLAREQLHVSVV 564 RCAAPQIRS RSLAAQEPASVLEEARLRLHVSAV 500 CHKQ SELEKAKATLLLATR 430 FSKVKKQLN SSYVKEEILKSANF 445 KNKESSSTSFLQDVKKKSDPHNNDFQSSLKEDQENYYINLLKNIKDPTDKAIRMMQLDVV 780 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 PKSLPCREREFENIYAFLEGKIQDQ--CGGCMYVSGVPGTGKTATVTGVIRTLQRMAKQN 622 PESLPCREQEFQDIYNFVESKLLDH--TGGCMYISGVPGTGKTATVHEVIRCLQQAAQAN 558 PKSLPCRSKEMEEITAFIKGSISDDQCLGRCMYIHGVPGTGKTISVLSVMKNLKAEVEAG 490 QDYLPARENEFASIYLSAYSAIESD--SATTIYVAGTPGVGKTLTVREVVKELLSSSAQR 503 PKYLPCREKEIKEVHGFLESGIKQSG-SNQILYISGMPGTGKTATVYSVIQLLQIKSRKK 839 ** * *..... :*: * **.***:* *.. * ELPAFEYLEINGMRLTEPRQAYVQIYKQLTGKT-VSWEQAHALLEKRFTTPA----PRRV 677 DVPPFQYIEVNGMKLTEPHQVYVQILQKLTGQK-ATANHAAELLAKQFCTRG----SPQE 613 SVSPYCFVEINGLKLASPENIYSVIYEGLSGHR-VGWKKALQSLNERFAEGKKIGKENEK 549 EIPDFLYVEINGLKMVKPTDCYETLWNKVSGER-LTWAASMESLEFYFKRVP---KNKKK 559 LLPSFNVFEINGMNVVHPNAAYQVFYKQLFNKKPPNALNSFKIIDRLFNKSQ---KDNRD 896. *: **:. :. * *... * -77 -

7 Results: ChapterI DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 TTVLLVDELDILCNRRQDVVYNLLDWPTKSAAKLVVVTIANTMDLPERLLMGKVTSRLGL 737 TTVLLVDELDLLWTHKQDIMYNLFDWPTHKEARLVVLAIANTMDLPERIMMNRVSSRLGL 673 PCILLIDELDVLVTRNQSVLYNILDWPTKPNSKLVVLGIANTMDLPEKLLP-RISSRMGI 608 TIVVLLDELDAMVTKSQDIMYNFFNWTTYENAKLIVIAVANTMDLPERQLGNKITSRIGF 619 VSILIIDEIDYLITKTQKVLFTLFDWPTKINSKLILIAISNTMDLPDRLIP-RCRSRLAF 955 : : : : **: *... * * *..... ::*::: ::******::. **:. : DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 DmORC1 HsORC1 AtORC1 ScORC1 PfORC1 DmORC1 HsORC1 AtORC1 ScORC1 pforc1 TRLTFQPYSHKQLQEIVTARLGGSE TRMCFQPYTYSQLQQILRSRLKHLK QRLCFGPYNHRQLQEIISTRLEGIN TRIMFTGYTHEELKNIIDLRLKGLNDSFFYVDTKTGNAILIDAAGNDTTVKQTLPEDVRK 679 GRLVFSPYKGDEIEKIIKERLENCK *: * * ---TFKGEAVQLVARKVAAVSGDARRALDICRRATEIADTAAVK AFEDDAIQLVARKVAALSGDARRCLDICRRATEICEFSQQKP AFEKTAIEFASRKVAAISGDARRALEICRRAAEVADYRLKKSN VRLRMSADAIEIASRKVASVSGDARRALKVCKRAAElAEKHYMAKHGYGYDGKTVIEDEN EIIDHTAIQLCARKVANVSGDIRKALQICRKAFENKRGHKIVPR *::: :**** :*** *:.*.:*::* * CVTMLHVQQALAEMIASAKVQAIRNCSR DSPGLVTIAHSMEAVDEMFSSSYITAIKNSSV ISAKSQLVlMADVEVAIQEMFQAPHIQVMKSVSK 710 EEQIYDDEDKDLIESNKAKDDNDDDDDNDGVQTVHITHVMKALNETLNSHVITFMTRLSF DITEATNQLFDSPLTNAINYLPW 1047 * MEQIFLQAlAAEVTRTGVEETTFMGVYQQVETlAAFM LEQSFLRAILAEFRRSGLEEATFQQIYSQHVALCRME LSRIFLTAMVHELYKTGMAETSFDRVATTVSSICLTN TAKLFIYALLNLMKKNGSQEQELGDIVDEIKLLIEVN AFKIFLTCLIIELRIINEFVIPYKKVVNRYKILIETSGKYIGMCSDNELFKIMLDKLVKM * GVTFPPPGRALRLCS KLGAERLIISEHSRNDLFQKILLNV 908 GLPYPTMSETMAVCS HLGSCRLLLVEPSRNDLLLRVRLNV 849 GEAFPGWDILLKIGC DLGECRIVLCEPGEKHRLQKLQLNF 787 GSNKFVMEIAKTLFQQGSDNISEQLRIISWDFVLNQLLDAGILFKQTMKNDRICCVKLNI 896 GILLIRPYIPLENISKNKSKEALLGFNESSKKGNNQKITRAQVSPDIDKESGDMGIELNV 1167 * ** SADDIHYALRVEEMVN SQDDVLYALKDE PSDDVAFALKDNKDLPWLANYL 809 SVEEAKRAMNEDETLRNL ETQLIITALMKDPDCSKKLNFY 1189 * : Figure 7: Comparison of the primary structure of ORC! from different organisms namely Dm, Drosophila melanogaster; Hs, Homo sapiens; At, Arabidopsis thaliana; Sc, Saccharomyces cerevisiae, Pf, Plasmodium falciparum. The amino acid sequence of ORC! from different organisms were aligned by CLUSTAL W multiple alignment program. C.) Shows weak: similarity between amino acids; C..) shows similar and C*) demonstrate identical amino acids

8 , Arabidopsis L-- Saccharomyces r Mus Xenopus L1L Homo lr su/folobus I... PfORCI Figure 8: Phylogenetic analysis of conserved PfORCl catalytic domain. Phylogenetic tree was made between PfORCl C-terminus and CdC6 sequences of Archaea and eukaryotes. PfORCl C-terminus shows maximum closeness with Archaea, Sulfolobus, ORClICDC6.

9 Results: ChapterI pforc 1 gene contains many important and diverse features and motifs as illustrated in Figure 9. It contains a putative PCNA binding motif which contains the signature motif QXX(I/L/M)XX(F IY)(F IY) at its C terminus and putative KEN box motif ~ E ru in middle of the protein. Both of these motifs are involved in degradation of replication proteins as described in other systems (Araki et al., 2006; Petersen et al., 2000). Primary structure analysis of pforc1 N-terminus domain shows many interesting and peculiar features. N-terminus of PfORC1 is unique in its amino acid composition, as it does not share any homology with any known protein among other Plasmodium species as well as in other systems. The N-terminus of pforc1 shows a putative leucine heptad repeats sequence (-LTNISSS-) that might be involved in DNA-protein or protein-protein interaction activities. A putative bipartite Nuclear Localization signal (NLS) is also located at the N-terminus suggesting this protein could be localized in the nucleus. The N-terminus also contains putative serine and threonine Cyclin-CDK phosphoryaltion sites (Figure 8) that might be used by Plasmodium Cyclin-dependent kinases (CDKs) to regulate pforc 1 activity during the different stages of P. falciparum development. 1.2 Homology modeling It is clear from the CLUSTAL W analysis that pforc1 holds maximum homology at its C-terminus as this part is more conserved in nature. Thus, in order to get an insight into the structure of the C-terminal conserved domain of pforc1, we decided to perform homology modeling based on the crystal structure available for S. pombe Cdc6 j Cdc 18. The crystal structure of Cdc6jCdc18 from yeast consists of 3 sub-domains [Liu et al., 2000]. First two domains (I and II) form an AAA+ type nucleotide- binding fold and third domain adopts a winged helix fold similar to known DNA-binding module of histones. The first two domains - 79-

10 Domain Illustration of PfORC I""'I"-r-... ~r-..., AA position Concensus sequence in PfORC1 * 0 0 = = = = Concensus Ser-, Thrphosphorylation sites Leucine heptad repeat Nuclear localization signal Putative KEN box 2-5 and TPKK/SPTK LTNISSS KKKKKKIKK KEN = Nucleotide binding domain Walker A-GXXGXGKT/S Walker 8-00/EXX D = Putative PCNA binding domain QKVLFTLF = Transmembrane domain Hydrophobic membrane binding domain Figure 9: Schematic representation ofpforcl. PfORCI ( amino acid residues) is shown as a schematic diagram where different functional domains with the respective amino acid residues and their concensus sequence are indicated in the figure.

11 Results: ChapterI of Cdc6/Cdc18 have 42% sequence homology with C-terminal part (amino acids ) of PfORCl. We have constructed a threedimensional model of PfORC1C ( ) using its Cdc6/Cdc18 homolog as a template, which exhibits a similar AAA+ type nucleotidebinding fold (Figure 10). This C-terminal part consists of two distinctive domains, domain I spans residues from 781 to 961 amino acids and domain II from 965 to 1033 residues. Domain I is a five-stranded parallel ~-sheet sandwiched by a pair of helices on both sides. This domain exhibits a Rossmann-type fold, commonly found in nucleotide-binding proteins [Erzberger and Berger, 2006]. Domain II is a small four Q-helix bundle connected to domain I by a coil (residues ). The putative nucleotide-binding site of ORC1, which is well conserved, consists of Walker A motif (GXXGXGKT/S) from 815 to 822 and the possible interaction between K821 with ADP ~-phosphate is shown in Figure 10. The Walker B motif DD/EXX is conserved between 903 and 906 residues. The extreme C-terminal end of PfORC1 (1090 to 1189) contains several hydrophobic residues including leucine and isoleucine that might form transmembrane domain(s). 1.3 Cloning, expression and purification of recombinant proteins to raise antibodies The ORF of PfORCl does not contain any introns within it. To clone the full-length gene of PfORCl, it was amplified from P. Jalciparum genomic DNA by specific primers P 9 and P10 (Table 4) as shown in figure 11A. 3.5 kb PCR product of PfORC1 was cloned into pet28a vector in BamHI restriction enzyme site. The recombinant clone was sequenced by using suitable overlapping primers as shown in table 4. For protein expression, E. coli BL21 codon plus cells were transformed using pet28a-pforcl. As PfORC 1 contains many repeat sequences (mostly asparagines and lysine) with codon biasness, it is difficult to express full length -140 kda protein in ample amounts in E. coli for the purpose of antibody generation. The - 80-

12 Figure lo:homology modelling of PfORCIC using the crystal structure of S. pombe Cdc6/Cdc18 as a template. The details of the modelling and the software used are described under Materials and methods. PfORC1 C consists of two distinctive domains, domain I spans residues from 781 to 961 amino acids and domain II from 965 to 1033 residues. Domain I is a five-stranded parallel ~ sheet sandwiched by a pair of helices on both sides. This domain exhibits a Rossmann type fold, commonly found in nucleotide-binding proteins. Domain II is a small four a-helix bundle connected to domain I by a coil (residues ). The putative nucleotide-binding site of PfORC1, which is well conserved, consists of Walker A motif and the lysine residue (K82l) interacts with ADP ~ - phosph a te. The extreme C-terminal part of PfORCl (1090 to 1189) is predicted to be a membrane bound domain and this domain is not included in the homology modelling...~

13 A. M 1 2 B. M(k His-PfORCl Da) In Anti-His Figure 11: Cloning and expression of PfORCl. (A) PCR amplification of PfORCl. 3.5 kb PfORCI gene was PCR amplified using specific primers as mentioned under Materials and methods,and the amplified product was subsequently resolved in 0.8% agarose gel. Lane I shows control PCR without genomic DNA template.the arrow indicates the position of the PCR product (lane 2). M stands for the molecular mass marker in kilo-base. (B) Expression of His-PfORCl. E.coli strain BL21 codon plus were transformed with pet 28a - PfORCl.The cell lysate from uninduced or induced bacterial cell pellet was analyzed by SDS-PAGE followed by Western blot analysis using anti-his 6 polyc1onal antibodies. A band of expected size was obtained only in the induced lane but not in the uninduced lane.

14 Results: ChapterI expression of full length pforc1 could be detected by western blot using anti-his antibodies (Figure 11B). However, the amount of protein was not enough to purify it to homogeneity. To raise specific antibodies against pforc 1, the C terminus (residues ) of pforc 1 (pforc 1 C) was amplified and subsequently cloned at the BamHI restriction endonuclease site of the pet28a vector and it was further confirmed by sequencing to rule out the possibility of any mutation during PCR. E. coli BL21 codon plus strain was transformed with this construct and bacterial culture was grown to an OD600 of 0.6. The culture was induced with 1 mm IPTG and grown further for 3 hr at 37 C. After induction, the protein was expressed but it was found to be in the pellet (inclusion bodies) due to its high hydrophobic nature. His6- pforc 1 was purified from the pellet by denatured purification using 1 % Sodium Lauryl Sarcosine in the lysis buffer as described in Materials and methods and it was purified by Ni-NTA affinity chromatography (Figure 12C). The purified protein was further confirmed as pforc1 by MALDI TOF analysis. This purified protein was used to generate polyclonal antibodies in rabbit and mice as described in Materials and methods. To generate polyclonal antibodies from another region of pforc1, we amplified the -546 bp region from extreme N-terminus part of pforc1 gene. The amplified gene was cloned at BamHI-NotI restriction site of pet28a vector. For protein expression and purification similar method was followed as described for His6-pfORC1C protein (Figure 12B). pforc1n protein was used to generate specific antibody in mice as described in Materials and methods

15 A PfORC1 ':::::::::=====:=:: PfORC1 C PfORC1 N B. ~ C. 66-"'W Figure 12: Expression and purification of different domains of PfORCl. (A) Schematic representation of full-length PfORC 1 and N-terminus and conserved C terminus regions of PfORC 1 that were used to raise antibodies against PfORC 1. The numbers indicate amino acid position. (B) Expression and purification of recombinant PfORCIN. BL21 codon plus cells were transformed with His6- PfORCIN. Recombinant plasmid and the cells were induced with O.5mM IPTG. Recombinant protein was solubilized from the inclusion bodies (pellet) with 1% sarcosyl and purified by Ni-NT A chromatography as described in Materials and methods. lane 1, Molecular mass marker (in kda); lane 2,uninduced cells; lane 3,induced cells; lane 4,supernatant; lane 5,pellet; lane 6, supernatant from solubilized pellet;and lane 7, purified protein. (C) Expression and purification of His6-PfORCIC terminus from inclusion bodies as described for PfORCIN terminus (B).

16 Results: ChapterI In order to compare the expression pattern of PfORC 1 with other replication initiation proteins during parasite development, we decided to raise polyclonal antibodies against two different Minichromosome Maintenance protein subunits namely, PfMCM4 and PfMCM7. As described earlier both MCM4 and MCM7 are the constituents of MCM 2-7 complex that is considered to be the helicase for fork movement (Tye and Sawyer, 2000). To raise antibodies against PfMCM4 (Accession no. PF130095), a part of the PfMCM4 ORF (nucleotide positions ; residues ) was cloned in the expression vector pet28a (Novagen) at the EcoRI restriction site and subsequently sequenced using suitable primers (Table 4). To purify the truncated His6-PfMCM4 fusion protein, Escherichia coli strain BL21 codon plus was transformed with pet28a recombinant plasmid construct. Truncated His6-PfMCM4 was purified by Ni-NTA (Qiagen) affinity chromatography under native conditions (Figure 13B). PfMCM4 protein specific antibodies were raised in two rabbits using standard protocol for antibody generation as described in Materials and methods. To raise antibodies against PfMCM7 (Accession no. PF ), a part of ORF (residues ) has been cloned into pet28a vector under BamHI- NotI restriction site. Protein was expressed and purified under native conditions using Ni-NTA (Qiagen) column chromatography (Figure 13D) as described under Materials and methods and further injected into mice to raise specific antibodies

17 A. Zn-finger motif PfMCM NTPase domain \..(-;;;;;==::;;;_ MCM signature motif,005 B M c. PfMCM7 1 MCM signature motif ) ( D Figure 13: (A) Schematic diagram of 1005 amino acid residues long PfMCM4. Different functional domains and their respective positions are marked. The solid black line (residues ) below indicates the region of PfMCM4 that have been expressed and purified for polyclonal antibodies generation in rabbit. (B) Expression and purification of PfMCM4 for the generation of polyclonal antibodies (described under Materials and methods). (C) Schematic diagram of PfMCM7. Amino acid position shows the MCM signature motif. Residues was cloned and expressed in E. coli BL21 codon plus strain to raise polyclonal antobody in mice. (D) Purification of PfMCM7 ( aa residues). Lane 'M' shows the molecular mass marker (kda).

18 Results: Chapter! 1.4 Antibody generation and characterization by western blotting using recombinant protein To investigate the specificity of antisera generated in rabbit and mouse, western blot analysis was performed using pre-immune and immune sera against the purified antigen (~ ng) namely PfORCIC, PfORCIN, PfMCM4 and PfMCM7. In all the cases pre-immune sera failed to recognize any band whereas the immune sera specifically recognized the purified protein (Figure 14 for PfORCl and Figure 15 for PfMCM4/PfMCM7, respectively)

19 A. Anti PfORC1 C PI I W.stfm Blot B. M(kDa) ,G,G G ~G ~;.p~~~ ~;.p ~~ ",~ < Western blot Commassie Antil!fORCIC c Commassie Western blot Anti-PfORCIN Figure 14: Characterization of PfORCI antibodies by western blotting. (A) Three hundred nanograms of recombinant PfORCIC protein was loaded in SDS-PAGE gel (shown as Commassie stained gel, bottom panel) followed by Western blot analysis using either anti-pforcl polyclonal antibodies or preimmune sera as control. (B) Western blot analysis to show that anti-pforcic antisera was unable to cross-react with MBP protein while it specifically reacted with purified recombinant His-PfORCl protein. (C) Specificity of anti PfORCIN antibody was teasted by western blot analysis using purified His PfORC 1 N (*) and MBP protein. Commassie stained gel showed the purified protein.

20 A. B. RecQrrbinant protein 'l ~ 33- Coomassie Westem tiot Westen blot Coomassie Stain Arti-PfMCMA Anti-PtMCM7 Figure 15: Specificity of PfMCM antibodies. (A) Three hundred nanograms of purified PtMCM4 (truncated) or Helicobacter pylori OnaB helicase protein were loaded in SOS-PAGE (as shown in Coomassie-stained gel) followed by Western blot analysis using anti-ptmcm4 antibodies. The antibodies crossreacted specifically with the recombinant PtMCM4 protein but not with the control HpDnaB protein. (B) Specificity of purified MCM7 protein was tested by wesern blot analysis using anti-ptmcm7 antibody.

21 Chapter II Modulation of PfORCl expression in Developmental stages of Plasmodium falciparum

22 Results: Chapter II 2.1 Stage specific expression of PfORCl and other replication initiation genes at the mrnalevel According to Li and Cox (2003), PfORC1 transcripts are present only in gametocyte stage while they are absent in asexual stages of the parasite. However, the transcriptome analysis of P. falciparum by Le Roch et al., 2003 using microarray analysis suggests that PfORC 1 gene is expressed both in asexual and sexual blood stages of the parasite. Due to the discrepancies regarding ORC 1 expression during developmental stages, we decided to find out independently the expression pattern of PfORC 1 in different developmental stages of intraerythrocytic developmental cycle of P. falciparum. In order to achieve our goal we analyzed the transcript level of PfORC 1 in different asexual stages using the semi-quantitative RT- PCR method. For this purpose, P. falciparum culture was first synchronized using the sorbitol method and parasites were harvested at the ring, trophozoite, and schizont stages. The total RNA was extracted from each synchronized stage using Trizol method and the cdna was made using the Stratagene kit essentially following the protocol described by manufacturer. Reverse-transcriptase PCR was done using gene specific primers set (as shown in table 4) with equal amount of cdna from all the three stages. Following PCR, the amplified products were resolved on 1.5 % agarose gel. In all the three stages we got a single band of expected size of around 600 bp suggesting that PfORC 1 is expressed at the mrna level during the asexual erythrocytic stages (Figure 16A). In order to rule out the possibility of genomic DNA contamination in the cdna preparation, total RNA was treated with DNase I and it was processed exactly the same way as it was done for the preparation of cdna, except for the addition of reverse transcriptase enzyme followed by PCR (-RT lanes). No product was obtained in these samples suggesting that the amplification of - 84-

23 A. AT + -+ Sl6 - Ring Troph, + filij.. l'!fl4}! Sch20m PfORG1 PfMCM4 PfMCMS PfGAPOti B. ~ c. Relative Abundance 10 Q) 8 Cl g 6 J:: o -0 4 ' DRing m Trophozoite Schizont IPfGAPD~ IPfORC1 mpfmcm4 opfmcm5 O~~~-.~~~~ g<!' ~.. #' ~$ <f-& ~ '" ~. Ring Trophozoite Schizont Figure 16: RT-PCR analysis of PfORCI and other replication proteins at the different erythrocytic stages. (A) RT-PCR analysis of PfORCI, PfMCM4, PfMCM5 and PfGAPDH using gene specific primers was performed using cdna obtained from the ring, trophozoite and schizont stages of the parasitic life cycle. PCR products were resolved in 1.5% agarose gel. RT (+) lanes indicates the PCR products that were obtained using cdna template generated using reverse transcriptase. No products were seen in the RT (-) lanes, suggesting that there was no DNA contamination in the RT products. (B) Fold change in the expression of PfORCI and other replication initiation genes at the mrna level during ring, trophozoite and schizont stages. The relative intensity of each band was calculated using densitometry scanning and the absolute values were obtained by normalizing against the background intensity. (C) Relative abundance ofpforci and other genes in different developmental stages of the parasite showed that these genes are maximally expressed in trophozoite and schizont stages.

24 Results: Chapter II PfORC1 specific products was obtained only from the cdna and not due to the genomic DNA contamination. In order to compare the expression pattern of PfORC 1 with other replication proteins at the transcript level, we performed the semiquantitative RT-PCR analysis of PiMCM4 and PiMCM5 genes using their specific primers under the same experimental conditions. As an internal control, the specific region of PfGAPDH, a house keeping gene, was also amplified under the same conditions. After resolving the amplified products on agarose gel, we found that the transcripts of PfORC1 and PiMCM4 were present in all the three stages and their expression levels peak at trophozoite and schizont stages while PiMCM5 transcript was present only in schizont stage. At the same time PfGAPDH transcript was almost similar in all the stages (Figure 16A). To compare the fold changes in the expression pattern of these genes in different developmental erythrocytic stages of P. jalciparum, we quantified the intensities of these amplified bands using densitometry scanning software and the absolute values were obtained by normalizing them against background intensity. These values were plotted graphically for three independent RT-PCR experiments (Figure 16B). These results indicate that the expression of PfORC 1 at mrna level goes upto four folds and eight folds from ring to trophozoite and schizont stages, respectively. The expression of PiMCM4 at the transcript level increases several folds during the later stages of developments. The expression level of PfGAPDH fluctuates only between 1 to 1.7 times from ring to schizont stages under the same experimental conditions. Simultaneously, to find out the relative abundance of these genes in different developmental stages, relative intensities of different bands at different stages for ORC 1 and MCM transcripts were compared with the

25 Results: Chapter II values obtained for control pfgapdh under the same experimental conditions. The results show that pforcl and PfMCM4 are expressed moderately in trophozoite stage and peaks during schizont stage. The expression of PfMCM5 is mostly detectable during schizont stage (Figure 16C). It is important to note that the majority of DNA replication in the parasites takes place during trophozoite to early schizont stages, therefore, the expression pattern of these genes coincide with the time of DNA replication. Further real time quantitative PCR will be required to estimate the exact fold increase of these transcripts at the later stages of the erythrocytic life cycle. 2.2 Stage specific characterization of PfORCl at protein level in different stages of P. /a/ciparum It is apparent from the RT -data that pforc 1 is expressed during intraerythrocytic stages in contrast to the data reported by Li and Cox (2003). To analyze the expression pattern of pforcl at the protein level during intraerythrocytic stages of the parasite life cycle, we used specific polyclonal antisera for western blot analysis using synchronized stagespecific parasite lysate. For this purpose, 3D7parasite culture was first synchronized with sorbitol method as mentioned under Materials and methods. Parasites at different developmental stages were harvested from early ring stage, late trophozoite, early schizont stage and late schizont stage. Tota1l cell lysate of different stages were resolved on 8% SDS-PAGE and transferred onto PVDF membrane for western blot analysis using antipforcic polyclonal antibodies. A single band of -150 kda was detected from trophozoite to mid schizont stages parasite lysate (Figure 17). The protein was not detected in early ring stage as well as in late schizont stage (46-48hr). To rule any possibility of cross-reaction from RBC - 86-

26 M(kOa) 212- : I 158-! 116- L.- ~~ Anti- PfORC1 Pre-Immune,t ~, ' ~,. ",. )t." -'." i ij' "',,. 1J.'i ~ ~' Coomassie stai ned gel Figure 17: Western blot analysis of PfORel in different developmental stages of P. Jalciparum. Equivalent parasite pellet from different stages were boiled in SDS PAGE loading buffer and proteins were resolved on 8% SDS-PAGE followed by western blot analysis using either anti-pforcl antibody or with pre-immune sera. A band of ~ 150kDa has been detected in immune lane during trophozoite and mid schizont stages but it was absent in early ring and late schizont stage. Under the same conditions uninfected RBC gave no detectable signal. Pre-immune sera failed to detect any signal at the same dilution in late trophozoite stage. Bottom panel shows the coomassie stained gel of the parasite extract to show the equivalent loading of the parasite proteins.

27 Results: Chapter II proteins, same amount of RBC pellet was also loaded in the western blot analysis. These results show that the -150 kda band of PfORCl is specific to the parasite protein and it is not due to cross-reaction with any RBC protein. In contrast to PfORCl, western blot using anti-pimcm4 antibodies detected a band of -120 kda under same conditions (Figure 18). These experiments also show the specificity of each antibody against their respective proteins. Furthermore, the absence of PfORCl at the late schizont stage compared to PiMCM4 clearly suggests differential regulation of these antibodies during development. In all the cases pre-immune sera did not react to any band under the same conditions confmning the specificity of these antibodies (Figure 17 and 18). Furthermore, the absence of PfORC1 at the late schizont stage compared to PiMCM4 clearly suggests differential regulation of these proteins during development. 2.3 Subcellular localization of PfORCl in asexual stages of P. Jalciparum In order to further determine the subcellular localization of ORCI in P. JaZciparum during erythrocytic stages, immunofluorescence assay was performed during different developmental stages of the parasite. It would also help us to confirm the status of PfORCl expression at the late schizont stage. For this purpose, thin smears were made on glass slides with synchronized culture of the parasite and they were fixed in prechilled acetone-methanol solution (90: 10 ratio). Finally, immunofluorescence assay was performed as described in Materials and methods using anti-pforc1c antibody or with pre-immune sera as primary antibodies followed by secondary antibodies conjugated with ALEXA Flour

28 A B. Anti - PfMCM4 Pre- Immune Coomassie stained gel Figure 18: Western blot analysis of PfMCM4. (A) Equivalent amount of parasite pellet from ring, trophozoite or schizont stages were boiled' in the SDS PAGE loading buffer followed by SDS-PAGE analysis and western blot analysis using either pre-immune or immune anti-pfmcm4 antisera. A band of -120 kda was detected in the immune lane but not in the pre-immune sera lane. (B) Coomassie stined gel showed the equivalent amount of parasite proteins that were loaded in all the lanes.

29 Results: Chapter II The signal for the presence of arc 1 was first visible during late ring/ early trophozoite stage ( hr) (Figure 19). A punctuate staining pattern was observed during mid-to-iate trophozoite stage parasites ( hr). This pattern continued till mid schizont stage ( hr). Surprisingly, during late schizont stage ( hr) the signal for ORC1 was completely abolished suggesting the absence of ORC1 during late stage of the erythrocytic developmental cycle. Immunofluorescence data nicely confirmed western blot analysis data that also showed the absence of PfORC 1 at the late schizont stage. These results collectively suggest that arc 1 is developmentally regulated in the P. Jalciparum. As a control, pre- immune sera used at the same dilution of the immunesera, failed to detect any significant signal under the same experimental conditions further confmning the specificity of the PfORC1C antibodies (Figure 19). In order to test the authencity of anti-pforc1c antibodies, we further performed immuno co-localization experiments using mid-to-iate trophozoite stage parasites with polyclonal antibodies raised against N terminus (anti-mouse) and C-terminus (anti-rabbit) of PfORC1 as primary antibodies followed by secondary antibodies conjugated to Alexa Flour-594 and Alexa Flour-488, respectively. The results indicate that both the antibodies show similar punctuate pattern nicely co-localized with each other (Figure 20). These results confirm the authencity of the antibodies raised in two different animals as well as the distinct punctuate pattern of PfORC 1 in the nuclei of P. Jalciparum

30 Alexa DAPI Flour 488 Merged Cell Early Ring EarlyTroph Mid Troph Late Troph Anti -PfORC1 (1 :4000) Mid Schizont Late Schizont Late Troph Pre -Immune (1 :4000) Figure 19: Subcellular localization of PfORC1 in asexual deve lopm~ntal stages of P. Jalciparum. Glass slides containing thin smears of P. falciparum-in fected erythrocytes from different synchronized stages were prepared and processed for immunofluorescence assay as described in Materials and methods. Green diffused signal of Alexa-488 for the presence of ORCl was observed during early trophozoite stage parasites. Distinct punctate staining was observed during late tarphozoite and mid schizont stage. Fluorescent signal of PfORCl expression was absent in early ring and late schizont stage. As a control, pre-immune sera was failed to give any signal in late trophozoite stage under the same experimental conditions. DAPI shows the staining of the nuclei. Bright field images are shown in 'Cell' panel.

31 DAPI Pf01C (Alexa 488) Pf01C (Alexa 594) Merged Merged 1 2 Cell Trophozoite Figure 20: Co-localization of PfORCl antibodies. C-terminus and N-terminus specific antibodies were raised in rabbit and mice, respectively and were used for the co-localization studies in trophozoite stage along with specific secondary antibodies (Goat anti-rabbit Alexa Flour 488 and Goat anti-mouse Alexa Flour 594, respectively) to ensure specifi city of the raised antibodies.

32 Results: Chapter II - Using immunofluorescence assay, we also determined the subcellular localization of other members of the pre-rc namely PfMCM4 and PfMCM7. Anti-PfMCM4 antibodies show diffused staining pattern during trophozoite and schizont stages completely merging with nuclear stain DAPI suggesting the nuclear presence of this protein in the nucleus all through different Plasmodium erythrocytic stages (Figure 21). In contrast, PfMCM7 is found predominantly in the nucleus during trophozoite stage where it is exported to the cytoplasm during late schizont stage (Figure 22) suggesting differential post-translational modification of these proteins. It is interesting to see different patterns of different members of the pre-rc following immunofluorescence analysis. While PfORC1 shows distinct punctate pattern, both the MCMs show diffused staining pattern. These results suggest different regulatory roles of these proteins for P. Jalciparum DNA replication control. 2.4 Expression of PfORCl in sexual developmental stages According to a recently published report, seventeen out of twenty five replication proteins are highly expressed in the gametocytes suggesting their role in sexual development of the parasites (LeRoch et al., 2003). To investigate the expression pattern of these replication initiation proteins in sexual blood stages, we performed immunofluorescence assay in the gametocytes. For this purpose, we harvested P. Jalciparum III-IV stage gametocytes and thin smears were made on the glass slides. The typical crescent-shape gametocytes were observed under microscope to ensure the gametocyte stages. Immunofluorescence assays were performed using either with mouse anti-pfg27 (a gametocyte-specific marker gene) (Lobo et al., 1999) or rabbit anti-pfnaps (a gametocytespecific nuclear protein) (Chandra et al., 2005), or anti-pfmcm4 or anti PfORC 1 antisera as primary antibodies and chicken anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibodies or - 89-

33 DAPI Alexa Flour 488 Merged Ring Troph Anti- PfMCM4 (1 :2000) Schizont Schizont P~PfMCM4 (1 :2000) Figure 21: Subcellular localization of PfMCM4 during asexual stages of P. Jalciparum. P. falciparum infected erythrocytes were treated for immunofluorescence assay using rabbit anti-mcm4 antibody as described in Materials and methods. Strong signal was found in trophozoite and schizont stage but no signal was detected in ring stage. Under the same conditions pre-immune sera gave no signal.

34 Alexa DAPI Flour 594 Merged Ring Troph Anti- PfMCM7 (1:1000) Schizont Schizont PI- PfMCM7 (1 :!OOO) Figure 22: Subcellular localization of PfMCM7 during asexual stages of P. Jalciparum. Different developmental asexual stages of P. falciparum were treated for immunofluorescence assay with anti-pfmcm7 at the dilution of 1: 1000 as primary antibodies and Alexa-594 as secondary antibodies. PfMCM7 expression was detected in all the stages while under the same conditions pre-immune sera gave no signal.

35 Results: Chapter II goat anti-rabbit ALEXA Flour 488 secondary antibodies (wherever appropriate). pfg27 showed distinct membrane localization pattern in the gametocytes as reported earlier (Lobo et al., 1999), whereas PfNAPS showed very strong nuclear signal as shown by Chandra et al. (2005). pforc1 showed strong nuclear staining that perfectly overlapped with the DAPI staining apart from some diffused staining in the cytoplasm. Interestingly, PrMCM4 showed a distinct punctate staining allover the gametocyte although the nuclear signal was more intense than the cytoplasmic signal (Figure 23). These results indicate that both pforc1 and PfMCM4 are also expressed during the sexual stage of the parasitic life cycle. It is interesting to note that both PfMCM4 and pforc1 are confined to the nucleus during the asexual stages, whereas these proteins are distributed in the cytoplasm as well as in the nuclei during sexual stage development. Their distinct patterns could be attributed to the gametocyte-specific function during gametocytogenesis, which can be explored further. 2.5 Immuno-gold electron microscopy Further, in order to reconfrrm the immunofluorescence data and to find out where in the nucleus pforc 1 is specifically located, immuno goldelectron-microscopic studies were perfonned with pforc1c antibodies using gold conjugated secondary antibodies. Parasites from early schizont stages were first fixed in a solution containing parafonnaldehyde and glutaraldehyde. Ultra thin sections were cut from the fixed blocks and further treated with primary (pforc1) and secondary (gold conjugated) antibodies as described in Materials and Methods and viewed under transmission electron microscope

36 DAPI Alexa Flour488 Merged Anti-Pfg27, I, J ; Anti-PfNAPS I '5f.:... -r..q#'- " ', Anti-PfORC I!.. ~'."",y'-:j(' I I,.' "'I'" I Anti-PfMCM4 Figure 23: Detection of PfORCl and PfMCM4 in sexual stage of P. Jalciparum. Gametocyte stages III-IV were used for the IFA studies. Affinity purified rabbit anti-pforc1 (1 :400) and affinity-purified rabbit anti-ptmcm4 (l :400) were used as primary antibodies. Mouse anti-pfg27 used as control for gametocyte stage-specific membrane protein at the dilution of 1 :750 and rabbit anti-pfnaps was used as marker for gametocyte stage-specific nuclear protein at the dilution of 1 :400. Goat anti-mouse Alexa Flour 488 and Goat anti-rabbit Alexa Flour 488 (Molecular probes) were used as secondary antibodies. DAPI was used as a marker for nuclear stain in each case. Slides were scanned under NIKON fluorescent microscope.

37 Results: Chapter II Immune sera gave distinct deposition of gold particles only in the parasite nuclei and very rarely in the cytoplasm, suggesting that pforcl is specifically located in the nucleus (Figure 24A). A larger view of the individual nucleus with pforcl signal is shown in Figure 24B (Nl from Figure 24A) or in Figure 24C (Nucleus from a different P. JaZciparuminfected RBC). pforcl is found to be present allover the nucleus in each case, suggesting that this protein is not confined to any particular subnuclear compartment. Pre-immune sera did not detect any specific signal in the nucleus under the same experimental conditions and antibody dilution, suggesting that the signals obtained from the immunoelectron microscopy are specific against pforc 1 only

38 Figure 24: Immllnoelectron-microscopic analysis of PfORCl localization in parasite-infected red blood cells. The secondary antibodies were conjugated to nm gold particles. (A) Parasite-infected RBC probed with anti-pforci antibodies. Three nuclei (N 1, N2. and N3) are shown in the frame. Arrowheads indicate the deposition of the gold particles within individual nucleus. (B) A higher resolution magnified version of N I from (A). Positions of the gold particle deposition and the nuclear membrane (M) are shown. (C) The pattern of the PfORCI distribution in an individual nucleus of a parasite-infected RBC (different from A). In each nucleus, (M) stands for the nuclear membrane; (P) stands for the parasite membrane; (R) stands for RBC and (N) stands for the nucleus. The scale bar is shown at the bottom right in each figure.

39 Chapter III Biochemical and junctional Characterization oj largest subunit ojorigin Recognition Complex, PjORCl

40 Results: Chapter III 3.1 Expression and purification of PfORCl C-terminus The genome of P. Jalciparum is highly AT-rich (~80%). Often the coding regions contain long stretches of asparagines and lysine repeats. The codon usage is different than of bacteria. It also contains low-complexity regions which could be attributed to the codon biasness nature of the parasite proteins. Therefore, it is extremely difficult to express Plasmodium proteins in heterologus system. Initially we cloned full-length PfORC1 (3.5 kb) in pet28a vector (Novagen) and checked its expression in E. coli BL21 Codon plus bacterial cells. However, we failed to express full-length protein in ample amounts for biochemical studies. We further decided to clone and express functional domains of PfORC1 for biochemical studies. As mentioned earlier, C terminus domain of PfORC1 is very well conserved and contains significant homology with ORC1 and Cdc6 counterparts from yeast to humans. C-terminus domain of PfORC1 contains conserved ATPase domain which consists of Walker A and Walker B signature motifs. As reported in yeast and humans, ATP binding and ATP hydrolysis activity of ORC 1. is essential for DNA replication initiation and origin activity (Bowers et al., 2004; Mendez and Stillman, 2003). It has been shown earlier that PfORC1 (C-terminal domain of PfORC aa) mostly goes into inclusion bodies when it is expressed in bacteria as a His-tagged fusion protein (Figure 12C). In order to get a soluble form of PfORC 1, that will facilitate biochemical experiments, we cloned PfORC1C in pmalc2x vector to get MBP-fusion protein. MBP tag often helps us to enhance the solubility of the protein. To further facilitate the solubility of the fusion protein, bacterial cells were induced at low temperature followed by purification of the fusion protein using amylose resin in native conditions as discussed in - 92-

41 Results: Chapter III Materials and methods (Figure 25B). A mutant form of the protein with a point mutation at the WALKER A ATP-binding domain [K to A] and MBP only were also purified under the same experimental conditions. Following protein purification and SDS-PAGE analysis of the purified protein, a major band of ~97 kda was found both in case of wild-type and mutant form of the protein. However, several smaller bands were also co-purified along with the major band, which are probably the degradation products of the upper major band (Figure 25B). 3.2 Nucleotide binding assay The C-terminal domain of PfORCl protein possesses conserved Walker A and Walker B domains. Walker-A domain contains G-X-X-G-X-G-K-T /S signature motif, where lysine is expected to interact with the nucleoside triphosphate tail. Thus, in order to test the nucleotide binding activity of PfORCl, we performed ATP binding assay. It is important to note that it has been difficult to obtain high yield of protein from MBP-PfORClC. Therefore, we decided to clone ~233aa of PfORC 1 C (residues from 694 to 899) in pmalc2x vector, termed as ~C-PfORCl that contained only Walker A-motif. This part of Pforcl gene is devoid of trans-membrane domain which could be responsible for the hydrophobic nature of the protein making it insoluble. Mutation in the ATP binding site of MBP-~CPfORCl (SGMPGTGKT) was introduced by PCR using the specific primers as listed in Table 4 by site-directed mutagenesis kit where lysine residue was converted into alanine. Wild-type and mutant MBP-~CPfORC 1 proteins were purified under native conditions (Figure 25D), as described under Materials and methods. For ATP binding assay, ~ 1 pg of the wild-type or the mutant MBP-~C PfORCl protein was incubated with radio-labelled (a-32pdatp) in ATP binding buffer and was further cross-linked with UV-irradiation as described in Materials and methods. Samples were resolved in lo% SDS

42 A C. ' c::::================j11 89 PfORC1 694c' ==:::::J*C:=:::::J, 1189 PfORC1 C / '\ 815-SGMPGTGKT -823 * SGMPGTGAT 694'-1 ---:*:0:: ,-1C-PfORCl D r 3, Figure 25: Expression and purification of MBP-PfORCIC-terminal domain. (A) Schematic diadgram of PfORCl Wild type (Wt) and PfORCIC-terminal domain. (*) shows the site of mutation generated in PfORCIC where lysine (aa position 822) was converted into alanine. (B) Purification ofmbp-pfolc Wild type and mutant protein. Alone MBP was also purified under the similar conditions. (C) Schematic diagram of delc PfORCl which contains only Waker A motif. (*) shows the site of genearted mutation where lysine of Walker A was converted into alanine as described in Materials and methods. (D) For Nucleotide binding assay MBP-,-1C PfORCl wild type and mutant proteins were purified under native conditions as described in Materials and methods.

43 Results: Chapter III PAGE and labeled protein was visualized in phosphorimager. Results showed that the wild type MBP-~CpfORCl protein was able to bind ATP but under the same conditions mutant MBP-~CpfORC 1 protein failed to bind ATP (Figure 26). 3.3 ATP hydrolysis assay pforcl protein is a member of AAA+ protein family (Bowers et al., 2004). The amino acid sequence of pforcl shows a putative NTP hydrolysis domain (WALKER B -DEID- at the position of aa) responsible for ATP hydrolysis. In order to find out whether MBP-pfORClC possesses ATPase activity, ATPase assay was performed using the purified MBPpfORClC protein. MBP alone was used as a control under the same experimental conditions. MBP-pfORClC showed ATPase activity as evidenced by the increasing trend of release of Pi (inorganic phosphate) with increasing protein amount whereas MBP alone protein did not show any ATPase activity under the same experimental conditions (Figure 27A). These results suggest that pforclc indeed contains ATP hydrolysis activity that might be important for origin activity in P. Jalciparum. ATP hydrolysis activity of pforclc protein was also measured in the presence of fixed amount of MBP-pfORClC protein but varying the time of incubation of ATPase reaction. The relative intensity of released inorganic phosphate (Pi) was quantified using densitometry scanning and the values were plotted graphically, as a function of time. The results indicate that the release of phosphate (Pi) increases as a function of time (Figure 27B)

44 Autorad I! Coomassie Figure 26: Nucleotide binding assay. Wild type and mutant ~C-PfORCl proteins were incubated with radiolabelled [a- 32 P]dATP and was further cross-linked using UV -irradiation, as described in Materials and Methods. Proteins were further resolved on 10% SDS-PAGE and labeled protein was visualized by phosphorimager. Bottom panel shows the presence of protein in both the lanes. (*) shows the position of ~C-PfORCl protein.

45 A. ~mp-prorcic MBP P I'oteiu \) \' '\- ~ ~ \'" (pmol) ~,..f " ~ Pi,~,t'.Up B. Time (min) ~mp-prorclc ~ \~ ~~ ~ ~ \,~,iiiji:: Figure 27: ATPase activity of MBP-PfORCIC. (A) ATPase assays were perfonned either using MBP-PfORCIC or MBP as described under materials and methods. Pi shows the released inorganic phosphate from the radiolabelled ATP with increasing amount of protein. The relative intensities of the spots were quantified by densitometry scanning and the results were plotted graphically was also plotted. (B) Time dependent ATPase activity of MBP-PfORCIC. The release of Pi at the different time points was assayed in the presence of 2 pmol MBP PfORCIC and quantitated by densitometry scanning and subsequently plotted graphically.

46 Results: Chapter III To further confinn that the ATPase activity associated with pforci protein was specifically related to pforci only and not due to any contaminating protein, increasing amount of a mutant fonn of PfORC 1 C, with a mutation in the WALKER A domain (K to A), was also used as control in the ATPase reaction along with the wild type protein. The homology model of pforcic also suggests that the lysine residue would be important for the binding to nucleotide triphosphate. The mutant protein failed to show any ATPase activity when compared to that of wildtype protein (Figure 28) even at the highest concentration of protein used (5 pmol) for ATPase activity. These observations further confinn the specificity of ATPase activity associated with pforci protein. 3.4 Functional characterization ofpforcl by yeast complementation analysis As discussed in Chapter I, pforc 1 shows 53% homology with ScORC 1. This homology is restricted to the C-tenninal region of pforci (pforcic) only. In order to detennine whether pforci is a true functional homolog of ORCI in vivo, we have perfonned genetic complementation assay in yeast using swapper strain of S. cerevisiae where the chromosomal copy of orc1 has been deleted and the wild type gene has been maintained in a plasmid for cell survival. For complementation assay, we made different constructs in yeast expression vector using either full-length pforci or ScORCI or different chimeras of yeast and Plasmodium arc 1. The schematic diagram of the constructs has been described in Figure 29. Details of constructs used for complementation: Initially all constructs used in this study were made in yeast expression vector prs416, containing a Galactose inducible promoter, tenninator sequence, multiple cloning sites (MCS) and ura gene as a selection

47 A. MBP-Pf01C Wt ~ MBP-Pf01 C Mutant ~ Protein (pmol) B.." :: 10 ~ ii: 5 20, MBP-PfORC1C W MBP-PfORC1 C Mutant Protein (pmol) Figure 28: ATPase assay of wild type and mutant PfORCIC. (A) ATPase activity of wild-type MBPORClC and mutant MBP-ORClC with increasing concentration of proteins (0 pmol to 5 pmol). (B) Spots corresponding to released Pi were quantified by densitometry scanning and the results were plotted graphically indicating the increasing trend of ATPase activity of PfORCI Wt with increasing protein amount while mutant ORCl protein failed to show any activity even at the highest concentration of protein used.

48 .. CONSTRUCTS ;2.01lIli Ifij... _.;ilwtrr il.lji1i1iiil'ik«&ulitbldmllgliirn:-" / / / SCORC1 PfORC1 SC-N-ORC1 Chimera I Chimera I Mutant Chimera II 1 454/ / ':;' Chimera III Figure 29: Schematic diagram of ScORCl, PfORCl and different chimera constructs used for yeast complementation. Shaded area in ScORCl and PfORCl shows the conserved part in both the proteins. (*) represent the site of mutation generated in Chimera I where lysine residue (822 aa position) of WALKER A motif was converted into alanine residue by site-directed mutagenesis.

49 Results: Chapter III marker. However, the swapper strain of yeast orcl, where orcl gene is maintained in an episomal plasmid, also uses ura as a selection marker. Therefore, after cloning into prs416 vector, the whole cassette containing galactose inducible promoter, the inserts containing full length gene or chimeras followed by terminator sequence was taken out using SacI-KpnJ restriction enzyme and the entire cassette was sub cloned into yeast expression vector prs314. This vector uses trp as a selection marker (Figure 30). Similar strategy was used for all the constructs. Finally, these constructs made in prs314 were used for complementation studies. Yeast orcl swapper strain was transformed with these constructs as described in Materials and methods using Lithium acetate method and the transformants were grown on plates containing SD-Trp media and incubated at 30 0 C for 3-4 days. Colonies on -Trp plates were simultaneously patched on -Trp plates and -Trp+5 FOA plates containing galactose and raffinose and incubated at 30 0 C for 3-4 days. Finally, patched colonies were further streaked onto -Trp and Trp+5 FOA (5 Fluoroorotic acid) plates for confirmation. 5 FOA used in this experiment is made into toxic compound and inhibits the growth of episomal plasmid containing wild type orcl gene under ura selection marker. Results from the complementation assay showed that all the clones grew happily on -Trp plates, whereas only ScORC1, Chimera I (containing ScORC1-N terminus and pforc1c), and Chimera III (Sc-N terminus, pforcl and ScORCl extreme C-terminus) survived in the presence of 5 FOA. All other constructs including full-length pforc 1 failed to survive in the presence of 5 FOA (Figure 31). To find out the importance of NTP binding domain of pforc1, we generated a point mutation in Walker A domain (K to A) of pforcl in - 96-

50 KpnI Terminator Kpnl prs416 Insert prs314 Ura SacI Trp prs314 Mod ified prs314 Figure 30: Illustration of yeast vectors used in yeast complementation assay. Vector prs416 contains ura gene as a selection marker, Gal promoter, terminator and multiple cloning sites (MCS) where insert was cloned under appropriate restriction sites as described under materials and methods. The entire cassette was digested with Sacl-KpnI restriction enzyme and subcloned in prs314 vector using the same restriction site in MCS. In vector prs314, trp gene is used as selection marker. Thus, the modified prs314 vector contains Gal promoter, terminator sequence and the chimera construct.

51 A. B Trp -Trp 1+5 FOA 4 Figure 31: Yeast complementation assay. Initially yeast orel swapper strain was transfonned with all the constructs, made for complementation assay, by Lithium acetate method as described under Materials and methods and plated onto SD-Trp medium. Transfonned colonies were streaked onto - Trp and - Trp/ +5 FOA plates (as shown in panel B). (1) ScORCl, (2) PfORCl, (3) Chimera I, (4) Chimera II, (5) Chimera III, (6) Sc-NORCl. All the constructs are in prs314 vector background.

52 Results: Chapter III Chimera I construct. This mutant Chimera I construct could not complement of yeast orc1 swapper strain in the presence of 5 FOA while the wild type Chimera I construct complemented functionally the same yeast strain under the same experimental conditions (Figure 32). To further confirm the results of complementation assay, we performed serial dilution test on -Trp and -Trp+5FOA plates using different dilutions of all the respective transformants selected previously on the similar plates. For this, serial dilutions (10 1 to 10-5 ) of yeast culture were made and further spotted on the plates followed by incubation at 30 0 C for 3-4 days. Spot test results showed the growth of ScORC1, Chimera I and Chimera IlIon -Trp+5 FOA plates where the growth of these constructs could be detected till 10-5 dilution. prs314 vector was used as a negative control (Figure 33A). Chimera I wild-type and Chimera I mutant were also tested for the viability in the serial dilution test where Chimera I wild-type was able to rescue the mutant yeast strain in the presence of 5 FOA at 10-5 dilution while the growth of Chimera I NTPmutant was severely affected in the presence of 5 FOA (Figure 33B) even in the undiluted samples. Thus the complementation analysis led to the two important findings: Firstly, it shows the functional evidence that pforc1 is a true homolog of orc1 in vivo. Secondly, the NTP-binding domain is essential for ORC1 function in vivo

53 Chimera I Mutant Chimera I - Trp - Trp/ +SF Figure 32: Functional importance of NTP-domain of PfORCl. Yeast complementation assay was performed using wild type and Mutant Chimera I. Mutation was generated in Walker A domain of PfORCI in Chimera I where lysine was converted into alanine as described in Materials and methods. Yeast orc J mutant swapper strain was transformed with wild type and mutant Chimera I construct and checked for their survival ability in -Trp and - Trp/ + 5 FOA plates. On - Trp plate, both the constructs were able to grow while in the presence of 5 FOA only wild type Chimera I was able to survive while mutant Chimera I failed to do so.

54 A. - Trp - Trp/ + FOA prs314 -SeORC1 prs314-pforc1 prs314-chimera I prs314 -Chimera II prs314 -Chimera III prs314- SeN ORC1 prs314 B. - Trp Chimera I wt Chimera I mutant -Trp I +FOA Chimera I wt Chimera I mutant Figure 33: Spot test assay..(a) To check the strength and survival ability of different constructs, ten fold serial dilutions were made and spotted onto - Trp and - Trp/ +5 FOA plates as described in Materials and methods. prs314 vector alone was taken as a negative control. (B) Spot test was performed for the wild type and mutant Chimera I. Wild-type Chimera I was able to rescue the mutant yeast strain in the presence of 5-FOA while mutant Chimera I failed to survive in the presence of 5- FOA.

55 Chapter IV Regulation of replication initiator protein PfORe1 by phosphorylation

56 Results: Chapter IV 4.1 Identification, expression and purification of putative phosphorylation domain in PfORCl Cell-cycle phases are difficult to describe in Plasmodium falciparum as nuclear division appears to be asynchronous during asexual blood stages (Inse1burg and Banyal, 1984). In eukaryotic systems an "ORC cycle" is described where differential regulation of ORC molecules is discussed in terms of replication and replication control mechanisms. Cyclindependent kinases (CDKs) are critical for the re-replication control that regulates the activities of components of pre-replication complex (pre-rc) after origin activation. In budding yeast, CDKs directly prevent pre-rc formation by inhibiting the function of the pre-rc components thus limiting pre-rc assembly to G 1 phase. CDKs appear to playa similar role in metazoans. Regulation of ORC activity is the premier step in determining the timing of DNA replication. Cell-cycle dependent changes occur in ORC activity as ORC tends to associate or dissociate from the chromatin. The "on and off' pattern of ORC molecules is referred as "ORC cycle" (DePamphilis, 2005). Regulating ORC association with chromatin is a feasible rnechanism for restricting re-initiation event in eukaryotes. In Yeast, phosphorylation plays a major role in regulating ORC activity. ScORC 1, ScORC2 and ScORC6 proteins are phosphorylated by Cyclin dependent protein kinase CDK1 (Cdc28)/Clb1 during G1-S transition and hyperphosphorylated during S-to-M phase transition (Nguyen et al., 2001). In mammalian cells also ORC 1 gets hyperphosphorylated by CyclinA/CDK1 at G2-M phase which prevents its stable association with chromatin and further leads to the absence of functional ORC-chromatin sites on metaphase chromatin (Li et al., 2004). It is very interesting to note here that in mammalian cells, three different states of ORC 1 are found during cell-cycle events. In G 1 phase ORC 1 associates with other - 98-

57 Results: Chapter IV ORC subunits during pre-rc formation, it is released from the chromatin and gets ubiquitinylate during S-phase and it is phosphorylate during M phase (DePamphilis, 2005). Thus phosphorylation could be a major controlling element which regulates activity of ORC subunits during cellcycle progression (DePamphilis, 2003; Saha et al., 2006). Apart from ORC components, Cdc6, MCM proteins and Cdtl are also post-translationally regulated by means of different mechanisms such as phosphorylation and ubiquitination (DePamphilis, 2003). Phosphorylation is driven by periodic activation and inactivation of Cyclin-dependent kinases (CDKs), which requires cognate Cyclin partners for their proper functioning. In order to search for the regulatory mechanisms associated with replication and cell-cycle control in Plasmodium Jalciparum, it is vital to look for the key regulatory molecules, which might give an important insight into the regulation of replication and cell-cycle analysis in P. Jalciparum. As it is evident from our western blot and immunofluorescence data, PfORC 1 is not detected during late schizont stage (Figure 17 and 19), whereas other replication proteins like PfMCM4 and PfMCM7 can be detected during late schizont stage. There is a possibility that ORCI is modulated in a cell-cycle dependent manner for the regulation of replication in P. Jalciparum. The amino acid sequence analysis of the largest subunit of Origin Recognition Complex, PfORC 1, reveals the presence of many peculiar and diverse features. As already discussed in Chapter I, extreme N- terminus of PfORC 1 contains putative Cyclin dependent kinase phosphorylation (CDK) sites containing consensus serine and threonine residues. In yeast, S. cerevisiae, and humans, ORCI gets phosphorylated in a cell - 99-

58 Results: Chapter IV cycle dependent manner for the regulation of replication (Li and DePamphilis, 2002; Mendez et al., 2002; DePamphilis, 2003). Thus, it will be quite interesting to find out whether these putative CDK phosphorylation sites at the N-terminus of PfORCI serve as the key targets for kinases leading to the modulation of this protein during the developmental cycle of Plasmodium in blood cells. The consensus sequence of CyclinjCDK phosphorylation site is (SIT) PX (K/R). In silico analysis of PfORCI sequence suggests the presence of two such putative sequences (2TPKKS and 2oSPTK23, respectively) at the N-terminus phosphorylation sites (Figure 34). Identification of these putative Cyclinj CDK sites will allow us to investigate whether PfORC 1 is truly phosphorylated in vitro and in vivo in combination with mammalian and Plasmodium CDK like kinases. Once we establish the phosphorylation event of PfORC 1 both in vitro and in vivo, its biological significance for the control of DNA replication will be revealed further. In order to find out the phosphorylation status of PfORCI in vitro, N terminus of PfORCI (aa position 1-164) was cloned in pgex6p2 vector between BamHI-NotI restriction sites. Protein was expressed in BL21 Codon plus cells and purified under native conditions as mentioned in Materials and methods. Different deletion derivatives (PfORC1-~Nl and PfORCl-~N2, deleting the first and second putative phosphorylation sites, respectively) were also cloned and purified under the similar conditions (Figure 35A). Purified wild type and deletion mutant derivatives of PfORCIN were used for subsequent In vitro phosphorylation studies

59 - TPKK - SPTK 1 \ / \/ 1189 PfORC1 1* * PfORC1N 1* * 1 (1-164) PfORC1-6 N11 ~ (6-164) PfORC1-6 N2 (25-164) MTPKKKIFQNFQANDNEILSPTKKGIKLNVSKLNILNFENTI ITKEKTNYEYKASLNKEIDEVLNNNNIINTHNNKKNNLNL Y DYNNIKNSTHEFYIDLNEQNKQTIKYNDNKFTPINKKEKYN LDETSSSSISSSL TNISSSL TNISSSL TNISSSLSNSLDE Figure 34: Schematic diagram showing the putative CDK phosphorylation sites in PfORC1.Extreme N-terminus of PfORCI possesses the conserved CyclinJCDK phosphorylation sites. (*) depicts the position of the phosphorylation sites. Bottom panel shows the amino acid sequence of the constructs used in the phosphorylation studies ofpfdrci. Putative CyclinJCDK sites are marked in bold letters

60 A B....- Nlb.tnt * CDK2 Figure 35: (A) Purification of PfORC1N and its deletion derivatives. Extreme N-terminus of PfORCl (PfDRCIN) and different deletion mutants (PfORC N 1 and.6. N2) were expressed and purified under the native conditions as described in Materials and methods. Proteins were resolved on 10% SDS-PAGE. (*) star shows the position ofpfdrcln and its deletion derivatives. (B) Co-purification of human CyclinAlCDK2 and CyclinE/CDK2 under native conditions as described in Materials and methods. (*) at higher molecular mass range shows the purified human CyA or CyE and (*) at lower molecular mass range shows the position of human CDK2.

61 Results: Chapter IV 4.2 Phosphorylation of putative kinase domains of PfORCl by human Cyclins/CDKs Based on sequence homology, PfORC1 contains putative conserved phosphorylation sites in its extreme N-terminus. Yeast and humans ORC1 as well as Cdc6 serve as a good substrate for S-phase dependent kinases like CyclinA/CDK2 and CyclinE/CDK2. Not much is known in Plasmodium regarding Cyclin/CDK partners and their possible targets. Thus, to find out whether PfORC 1 serves as a substrate for human Cyclins/CDKs, human CyclinA/CDK2 and human CyclinE/CDK2 were expressed and co-purified as described in Materials and methods. Copurification of hcyclina/cdk2 or hcycline/cdk2 is essential as these Cyclins binds to catalytic cleft at one side of CDK2 and induces conformational change resulting in the active Cyclin/CDK complex (Figure 35B). To confirm the activity of bacterially purified hcyclin/ CDK2 complex, we performed in vitro kinase reactions using Histone HI as a substrate in the presence of above enzymes. After the reaction, products were resolved in 10% SDS-PAGE followed by autoradiography. Both hcyclina/cdk2 as well as hcycline/cdk2 can phosphorylate Histone HI (as shown in the Figure 36) suggesting that these enzyme complexes are active under these experimental conditions. Coomassie stained gel shows the presence of equal amount of proteins in each case. In order to investigate whether putative Cyclin-CDK phosphorylation sites present in PfORC1N, can be phosphorylated in vitro, kinase reaction was carried out using purified recombinant GST-PfORC1N protein and its deletion mutant derivatives as described in materials and methods. GST-PfORC1N showed an intense signal of phosphorylation in the presence of hcyclina/cdk2 and radiolabelled y_32p ATP, while PfORC1- ~N 1 showed much reduced phosphorylation status in comparison to the

62 32 - P Histone Hi Coomassie Figure 36: Phosphorylation of Histone HI by human CyclinAlCDK2 and CyclinE/CDK2. Kinase assay was performed with bacterially purified human CyAlCDK2 and CyE/CDK2 using Histone HI as a substrate as d.escribed in Materials and methods.upper panel shows the phosphorylation of Histone HI while lower panel shows the coomassie stained gel to show the presence of Histone protein.

63 Results: Chapter IV - wild type protein under the same experimental conditions. PfORC 1-~N2 did not show any phosphorylation under the same experimental conditions (Figure 37A). Similar results were obtained using hcyc1inejcdk2 (Figure 37B) as kinase enzymes. These results confirm that the putative Cyc1in-CDK phosphorylation sites can be phosphorylated using active Cyc1in-CDK enzyme. These results also confirm that the phosphorylation of extreme N-terminal domain of PfORCl is specific since deletion of both the Cyc1in-CDK sites completely abrogates phosphorylation events. 4.3 Biochemical characterization of PfORCl kinase domain by hcyclinalcdk2 We have shown that that PfORCl is phosphorylated by hcyc1insjcdks and the sequential deletion of one or two phosphorylation motifs from N terminus results in the reduction or complete loss of phosphorylation status, respectively. To further investigate thoroughly hcyc1injcdk dependent phosphorylation of PfORCIN, we performed time dependent, enzyme concentration dependent and substrate concentration dependent In vitro kinase reactions using recombinant GST-PfORCIN and hcyc1inaj CDK2 proteins. We find that phosphorylation of PfORCIN increases in a time dependent manner confirming that the in vitro kinase activity follows a time dependent kinetics (Figure 38A). Coomassie stained gel confirmed the equal loading of Cyc1inA and GST-PfORCIN proteins. Further we investigated substrate dependent phosphorylation of PfORCIN in the presence of equal amount of kinase enzyme. Phosphorylation of PfORC 1 increased with increasing concentration of protein amount (Figure 38B). Finally, we determined the dependence of PfORCIN phosphorylation on enzyme concentration. We find that increasing concentration of

64 A. CyclinAlCDK2 Autorad Coomassie CyclinE/CDK2 B. Autorad Coomassle Figure 37: Phosphorylation of PfORCl by human Cyclins/CDKs. (A) Kinase assay was perfonned using human CyclinAlCDK2 (0.25 Ilg) as enzyme and GST-PfDRCIN (0.5 Ilg) and its deletion mutant derivative as substrates. Upper panel shows the coomassie stained gel containing all the proteins. (B) Kinase assay was perfonned using human Cyelin E/CDK2 as enzyme and GST-PfDRCIN or GST-PfDRClb..N2 as substrates.

65 A. J 25 Time (min) 'ti CyclinAlCdk ~ 20 ~ 32 P.PfORC1 N :,M Autorad Coomassie i 15.b GI 'tv 10 ~ a. III o 5 ~ Il. o Time (min) B. J Substrate (PfORC1N)(ng) CyclinAlCdk P.PfORC1 N Autorad Coomassie 35 'i 30.. of 25 ~ E 20..! 15 IV i 10 8 f 5 or-~--~--~--~~--~ o Substate Concen.(ng) Figure 38: (A) Time dependent phosphorylation of PfORCIN by human CyclinNCDK2. Kinase assay was performed at different time point with human CyclinNCDK2 (0.25 ~g) and GST PfORCIN (0.5 ~g) as a substrate. Bottom panel shows the equal loading ofcyclina as well as GST-PfORCIN in all the lanes. Band intensities were calculated by densitometry and values were plotted graphically. (B) Substrate dependent phosphorylation of PfORCIN by human CyAlCdk2. Kinase assay was performed with increasing concentration ofpforcln. Bottom panel shows the coomassie stained gel with increasing amount of GST-PfORCIN. Phosphorylated population of PfORCIN was measured by densitometry and values were plotted graphically.

66 Results: Chapter IV hcyclina/cdk2 results In the increased phosphorylation of GSTpfORCIN (Figure 39). Collectively, all these results further confirm that the Ser, Thr residues within the putative CDK phosphorylation sites at the N-terminus of pforcl can be phosphorylated in the presence of hcyclins/cdks. These results also indicate that these sites can be potentially phosphorylated by the putative CDK like kinases in P. falciparum. 4.4 In silico screening of Plasmodium kinases and eyclins Cell division cycle in P. falciparum is different from fundamental processes of cell division as found in eukaryotes. In Plasmodium, celldivision is asynchronous and multiple rounds of nuclear division take place without the dissolution of nuclear membrane. Due to this asynchrony, cell-cycle stages are also not defined and thus there is no clear demarcation between G 1, S, G2 and M phases of the cell-cycle. In Plasmodium falciparum, different CDKs and Cyclins (cell-cycle control elements) have been identified either by PCR method using the degenerate primers or using the BLAST search analysis using known CDKs and Cyclins as queries. Although several CDK like kinases have been reported in Plasmodium, no substrate for CDK like kinases has been assigned yet. Although the functional role of pfcyclins and CDKs in cell-proliferation is not known, their putative function in cell-multiplication can be suggested on the basis of expression profiling during developmental stages provided by micro array data (Bozdech et al, 2003; LeRoch et al, 2003). A representation of the expression profiling of CDK-related protein kinases and pfcyclins during the erythrocytic schizogony is shown in figure

67 CyciinAlCDK2 (/19) ] PfORC1N P-PfORC1N Coomassie PfORC1N Figure 39: Enzyme dependent kinase assay of PfORCl by human CyclinAlCDK2. In vitro kinase assay using GST PfDRCIN and increasing concentration of human CyAlCDK2 in the presence of y _ 32 PATP. Upper panel shows the phosphory -lated population ofpfdrcl and lower panel shows the cooma- -ssie stained gel to show the equal amount oforcl protein in all the lanes.

68 F:xprcssion prior 10.Ild - durin!,! So-!H'aase Exp~oadariDg~pb~ -.ad DucJ~r division;---- Exp... lod during lau Kblzuo;.r..., - No stro;'g expression pattern appa... nt CDKJ cyclins I J. PtcK2 subunits I:~ ~.~ PtCK2e' 'M5!~iAJ... ~ of... _ --PtCK2R '~.. T >:: t:':.. ; '':''::: 1>1CK2RJ Figure 40: Representation of expression profile of CDK- related protein kinases and cyclins (adapted from Sherman, I.W., 2005)

69 Results: Chapter IV The expression profile results suggest that PWK5, PfCrk-l, PfCyclin4, pfcyclin2 and PfCrk-5 are mainly expressed during S-phase and nuclear division cycle. The putative cell-cycle control elements including CDKrelated kinases and Cyclins are shown in figure 41. It also depicts the possible downstream targets that mainly include pre-rc members. Nothing is known about the targets and mechanistic importance of CDKrelated kinases and Cyclins involved in cell proliferation in Plasmodium. Since pforc 1 is a replication initiation protein, we have hypothesized that it could be a potential target for S-phase kinases and Cyclins. It will be interesting to study the identification and biochemical analysis of putative CDKs and their cognate Cyclin partners involved in phosphorylation of pforc 1 that may lead to the regulation of ORC function and cell cycle control in parasites. In our study we have used different CDKs and Cyclins combination to study the phosphorylation status of pforcl in vitro. Table 9 shows the main features of Plasmodium kinases and Cyclins used in our study

70 Putative cell cycle control elements I CDK-related kinases - * PfPK5 : MAL13P PfPK6 : PF * Pfcrk-l : PFD0865c - Pfcrk -3 : PFD0740w - * Pfcrk-4 : PFC0755c - Pfcrk-5: MAL6Pl Pfmrk : PFIO 0141 Cyclins -* Pfcyc-l : PF14_ Pfcyc-2: PFL1330c -* Pfcyc-3 : PFE0920c - Pfcyc-4: PF13_ Control of entry into S-phase - 3 ORC homologues: PfORC1, PfORC2 and putative PfORC5-7 MCM homologues : PfMCM2, PfMCM3, PfMCM4, PfMCM5 PfMCM6, PfMCM7 and -Replication proteina : PFI0235w -RFC subunit: PF A0545c Figure 41: Putative cell cycle related proteins in Plasmodiumfalciparum. CDK-related kinases and cyclins with their PlasmoDB identifier are listed. The potential targets/substrates (mainly pre-rc members) for Cdk related kinases are also shown. (*) shows the S-phase kinase and cyclins in P. Jalciparum.

71 Results: Chapter IV Table9: Brief description ofthe kinases and Cyclins involved in the study Comments Reference 1. PtPK5 2. PtPK6 3. PfCRK4 4. PfCRK5 5. PfCyc-1 6. PfCyc-2 andpfcyc-4 7. PfCyc-3 8. RINGO First CDK related kinase which shows 60% Ward et ai., 2004 homology with CDK1 and CDK5 of eukaryotes. Activity peaks at schizont stage (36hr post Merckx et ai., 2003 invasion). Phosphorylate Histone HI from Plasmodium LeRoch et ai., 2000 extract Autophosphorylate in the presence ofpfcyc-1 Homologus with CDKs and MAPKs. Cyelin-independent kinase. Peaks in trophozoite and schizont stage Localized in nueleus and cytoplasm Related to CDKs and MAPKs. Weak Histone HI phosphorylation activity ill immunoprecipitation experiment from Plasmodium extract Associated with CDK1I2/3 group. Phosphorylate Histone HI in immunoprecipitation experiment in Plasmodium extract No Cyelin partner is known yet. Present in parasite prior to S-phase Phosphorylate Histone HI from Plasmodium extract as shown by pull-dowm and immunoprecipitation experiment Present in late trophozoite and schizont stage Associated with kinase activity of Histone HI in parasite extract No kinase subunit is known Miyata et ai., 1999 Bracchi-Ricard et ai., 2000 Equinet, L., et ai. (unpublished data). Nivez, M.P. et ai. (unpublished data) Merckx et ai., 2003 Merckx et ai., 2003 Functional homo logs of G lis-phase Cyelin D or Merckx et ai., 2003 E Activate PtPK5 Associate with kinase activity of Histone HI from parasite extract in immunoprecipitation No sequence similarity with Cyelins Merckx et ai., 2003 Bind and activate Cdc2 or CDK2 in vitro Required for G21M phase transition during oocyte maturation in Xenopus Shown to be efficient activator ofptpk5 in in vitro kinase reactions

72 Results: Chapter IV 4.5 Expression and purification of PfCDK-related kinases and Cyelins To study pforc 1 phosphorylation in vitro, different Plasmodium CDKs and Cyclins were expressed and purified under native conditions as discussed in Materials and methods. These constructs were obtained from Prof. Chritian Doerig (University of Glasgow) as kind gifts. Both pfpk5 (~28 kda) and pfpk6 (~35 kda) were purified as His6- tagged proteins. pfcrk4 (~66 kda) and pfcrk5 (~70 kda) kinases were purified as GST-tagged proteins. pfcyc1 (~52 kda), pfcyc2 (~56kDa), pfcyc4 (~56kDa) proteins were expressed and purified as GST tagged fusion proteins whereas pfcyc3 (~62 kda) was purified as MBP fusion protein. We also purified an activator of pfpk5, RINGO as a MBP-tagged fusion protein. RINGO has been shown previously as an activator for pfpk5 function (Merckx et al., 2003). The purification of all the above proteins is shown in Figure 42. The major band corresponding to each purified protein is indicated by asterisk (*). The molecular mass of the fusion proteins as shown after SDS-PAGE analysis corresponds with their theoretical molecular mass. These purified proteins were used for in vitro phosphorylation assay. 4.6 In vitro kinase assay of PfORCl with Plasmodium kinases and PfCyclins in vitro phosphorylation of PfORCIN using purified PfPK6 and different PfCyclins: pfpk6 was first isolated as a novel protein kinase in P. Jalciparum (Miyata et al., 1999).The catalytic domain of pfpk6 shows 57.3% homology with Cyclin dependent kinase (CDK) and 49.6% homology with mitogenactivated protein kinase (MAPK). pfpk6 is predominantly expressed during trophozoite and schizont stages. It is localized both in the nucleus as well as in the cytoplasm during developmental stages. pfpk6 can be

73 Figure 42 : Purification of CDK-related kinases and cyclins of Plasmodium Jalciparum. Different Plasmodium kinases and Cyclins were expressed and purified under native conditions as described in Materials and methods. RINGO (Xenopus origin) is an activator of cdc2-related kinases and it can stimulate PfPK5. It was expressed and purified under native conditions as MBP-fusion protein. (*) shows the position of respective recombinant protein.

74 Results: Chapter IV auto-phosphorylated and it can phosphorylate other substrates like HistoneHI and ribonucleotide reductase in the absence of a Cyclin partner (Bracchi-Ricard et al., 2000). In an effort to test the ability of PfPK6 as a kinase in in vitro phosphorylation studies, we performed kinase reaction in the presence and absence of Histone HI as a substrate as described in Materials and methods. The recombinant bacterially expressed PfPK6 can phosphorylate histone as well as itself (Figure 43A). These results confirm that PfPK6 is active in in vitro kinase reaction. Further, to characterize whether PfPK6 can use pforcl as a substrate or not, we performed kinase reaction using recombinant PfPK6 and all four pfcyclins (pfcyc1ins 1-4). The results indicate that PfPK6 can phosphorylate pforc IN only in combination with pfcyc-2 and pfcyc-4 although it is autophosphorylated in all four cases suggesting that substrate specificity of PfPK6 may depend on its right Cyclin partner (Figure 43B) Phosphorylation of PfORCl by PfCRK5 and PfCyclins combination In order to screen further P. Jalciparum kinases as potential modulator for pforcl phosphorylation activity, we used another S- phase specific kinase, pfcrk5. pfcrk5 is related to the CDKlj2j3 group of kinases that also shows homology with PfPK6 (Nivez, M.P. et al. (unpublished data). Immunoprecipitation of PfPK5 from asexual stage parasite extract shows Histone HI phosphorylation activity. Nothing is known about its Cyclin partners as well as its potential targets in the Plasmodium Jalciparum

75 A. PfPK6 Histone HI ~, '~ _-:~ ~ ~ -ii ~ ~ - _ ~ r' \ ), ~,.~.,(1 I }, j, ~ ~ I, -b"l'< >:~~~~._~, ~ m=-"=~ _~~.~_ i Autorad Coomassie B. PfPK6 Autorad Coomassie PfORC1N PfPK6 PfORC1N PfPK6 Figure 43: (A) Autophosphorylation and in vitro kinase activity ofhis-ptpk6 was assayed in a standard kinase reaction using His PtPK6 (-1 ~g) alone (lane 1) or His-PtPK6 (-1 ~g) along with Histone HI (lane 2) or Histone HI (lane 3) in the presence ofy}2 P ATP. (B) In vitro kinase assay using His-PtPK6 (- 600 ng) and GST PfDRCIN (- 800 ng) as a substrate in combination with PfCycl, PfCyc2, PfCyc3 and PfCyc4. Coomassie stained gel shows the equal loading of the GST-PfDRCIN and His-PtPK6 in all the lanes.

76 Results: Chapter IV Initially we tested the activity of PfPKS in combination with pfcyclins using Histone HI as a substrate. The results indicate that only PfCyc-l and pfcyc-4 can phosphorylate Histone HI. As it is clear from the Figure 44A, that only pfcyc-l and pfcyc-4 phosphorylate HistoneHI. The results suggest that pfcyc-l and pfcyc-4 are the possible Cyclin partners to make it an active kinase for Histone HI substrate. Further, to find out whether pforcl can serve as a substrate for pfcrk5, we performed kinase reaction in the presence of all four pfcyclins using GST-pfORCl as a substrate. As it is evident from Figure 44B, pfcrk5jpfcyclin combination fails to phosphorylate pforcl Phosphorylation of PfORCl by PfPkS and PfCyclins / RINGO pfpks was identified as a first protein kinase related with CDKl and CDKS family of kinases. It shows maximum homology (60%) with CDKl homo logs of higher eukaryotes. Although it shows Histone phosphorylation activity following immunoprecipitation from Plasmodium extract, recombinant pfpks does not show any histone phosphorylation activity in the absence of Cyclin partner. Monomer of pfpk5 is also not an active form of the enzyme. pfpk5 activity can be stimulated in the presence of pfcyc-l, human Cyclin H and RINGO (an activator of CDK2 in vertebrates) (Merckx, A. et al, 2003). So far Histone HI is the only substrate known for pfpk5 activity. In order to check whether recombinant pfpks is active or not in our experimental conditions, we performed kinase reaction in the presence of all four pfcyclins as well as RINGO using Histone HI as a substrate. We find that pfpk5 can phosphorylate Histone HI only in the presence of RINGO (Figure 45). On the contrary, we could not able to detect any phosphorylation of Histone HI in the presence of pfcyclins. Previously it

77 A. PfCRK5 32 -P Histone H1 Coomassie B. PfCRK5 Autoradiogram PfORC1N Coomassie PfORC1N Figure 44: Phosphorylation of Histone HIJPfORCl substrate by PfCRK5. (A) Kinase assay was performed to check the Histone HI phosphorylation activity of PfCRK5 in combination with all four PfCyc1ins or RINGO. Coomassie stained gel showed the loading of equal amount of protein in all the lanes. (B) In vitro kinase assay was performed to check the phosphorylation status ofpforci by PfCRK5 and four PfCyc1ins as described in Materials and methods. Bottom panel showed the coomassie stained gel as a loading control.

78 PfPK5 32 P-Histone HI Autorad Histone HI Coomassie Figure 45: Activation of PfPKS by RINGO. Standard kinase assay was perfonned with PfPK5 in combination with all four PtCyclins or MBP-RINGO using Histone HI as a substrate. PfPK5 was able to phosphoryate Histone HI only in the presence of RINGO protein. Upper panel shows the autoradiogram following kinase reaction. Coomassie stained gel (lower panel) shows the equal amount of Histone HI in all the lanes.

79 Results: Chapter IV was reported that PfPK5 autophosphorylates in the presence of pfcyc-l (LeRoch et al., 2000) but in our experimental conditions we were unable to detect autophosphorylation of recombinant PfPK5 in the presence of pfcyc-l. These results suggest that PfPK5 is active only in combination with RINGO. Further, in order to test its ability to use PfORCl as a substrate, we performed the kinase reaction in the presence of GST-pfORC1N as a substrate. It is clear from the Figure 46A that GST-pfORC1N is phosphorylated by PfPK5 only in combination with RINGO protein. In order to find out that whether this phosphorylation event is specific to the Cyclin-CDK phosphorylation site identified at the N-terminal region of pforc1, we performed kinase assay using GST-pfORC1N and GSTpfORC1~N2 as substrates. We find that PfPKS and RINGO combination can phosphorylate wild type but not mutant pforc1n suggesting the specificity of PfPKS for CDK phosphorylation sites (Figure 46B). 4.7 Effect of CDK inhibitor, Roscovitine, on PtPK5 activity Substrate specificity of phosphorylation events by their kinase/cyclin complexes can be studied by specific inhibitors. As evident from the literature roscovitine is a potent CDK1/2/S inhibitor. Previously, Roscovitine has been used in Plasmodium to study the inhibitory effect of autophosphorylation of PfPK6, where it was shown that it can specifically inhibit PfPK6 autophosphorylation activity at a concentration of M in the culture. We have found that PfPKS IS a CDK1 related kinase that can phosphorylate histone HI as well as pforc1n. Further we were interested to find out the effect of cdc-2 related kinase inhibitor, roscovitine, on PfPKS kinase activity on pforc1 substrate

80 A. PfPK5 32.p PfORC1 N Autorad PfORC1N Coomassie B. PfPK5 / Ringo Autorad Coomassie Figure 46 : Phosphorylation of PfORCIN by PtPK5. (A) In vitro kinase assay was perfonned by adding recombinant proteins. (His PtPKS in combination with PfCyclins or RINGO) in a reaction mixture containing GST-PfDRCIN as a substrate. Top panel shows the autoradio -graph and lower panel shows the coomassie stained gel as a loading control. (B) Kinase assay was perfonned with PfPKS and RINGO using PfDRCIN or PfDRCl A N2 as substrates. (*) shows the band of phosphorylated PfDRCIN in autoradiograph. Bottom panel (coomassie stained gel) shows the equal amount of loading oforci proteins.

81 Results: Chapter IV For this purpose, we performed kinase reaction in the presence of increasing concentration of roscovitine. PfPK5 can phosphorylate GSTpfORCIN in the absence of roscovitine (Figure 47A). However, the kinase activity of PfPK5 is inhibited in the presence of roscovitine. It is interesting to note here that at a concentration ofloo pm, there is a sharp decrease in the phosphorylation of pforcin. To check the inhibitory effect of roscovitine on the phosphorylation activity of PfPK5, we measured the intensity of phosphorylated pforcin band by densitometry scanning and plotted the values graphically against the increasing concentration of roscovitine (Figure 47B). This graph clearly shows that roscovitine very effectively inhibits the PfPK5 phosphorylation activity suggesting that PfPK5 is responsible for pforc 1 phosphorylation. 4.8 Pull down of P. Jalciparum kinases from Plasmodium extract followed by kinase assay ofpforcl In vitro kinase assay results indicate that PfPK6 phosphorylates pforc 1 in combination with pfcyc-2 and pfcyc-4 and PfPK5 phosphorylates pforcin in combination with RINGO protein as a Cyclin partner. These results suggest that there is specificity for pfcyclin/pfcdk combination for the activation of these kinases. Alternatively, it is possible that pfcyclins used in this study may have unidentified kinase partners that may lead to the formation of active pfcyclin/pfcdk complex in P. Jalciparum. In order to find out whether pfcyc1ins are capable of capturing cognate pfcdks from P. Jalciparum extract leading to the formation of active pfcyclin/pfcdks complex, we performed pull-down assay using GST or MBP bead containing pfcyclins as fusion protein and P. Jalciparum extract from trophozoite and schizont stages followed by kinase assay

82 A. ==========-]1 Roscovitine o (IlM) PfORC1N Autoradiogram PfORC1N Coomassie B. 'P'hOSfi~b:ate transffer@d -4- Phosp,hat'E! tcransl'ereld o ton mm Rosc,o,vitinle CEJlflC. (um) Figure 47: Effect of CDK inhibitor, Roscovitine, on phosphorylation of PfORCl. (A) In vitro kinase assay was perfonned with PfPKS, RINGO and GST-PfDRCIN in the presence of increasing concentration of CDK inhibitor, roscovitine. Lane 1 shows the phosphorylation ofpfdrcin in the absence ofroscovitine and lanes 2-5 show the phosphorylation ofpfdrci at increasing concentration ofroscovitine. Bottom panel shows the coomassie stained gel with equal loading of GST-PfDRCIN in all the lanes. (B) To obtain a graphical representation, band intensities were quantified using densitometry scanning and plotted against the increasing concentration of roscovitine.

83 Results: Chapter IV using either PfORCIN or Histone HI as substrates. The details of pulldown experiments are discussed in Materials and methods. The pull-down experiment indicate that all the pfcyclins can phosphorylate Histone HI as a substrate using Plasmodium extract obtained from either trophozoite and schizont stage parasites suggesting formation of active kinase complex (Figure 48A and 48B). In comparison to the phosphorylation of Histone HI, phosphorylation of pforc I was not very significant following pull-down experiments under the same experimental conditions. Although wild type pforcin but not mutant pforcin showed weak phosphorylation in the presence of pfcyc-1 and pfcyc-2 but pfcyc-3 and pfcyc-4 failed to show any phosphorylation activity using WtpfORCIN as a substrate following pulldown experiments (Figure 48C and 48D). 4.9 Pull-down of PfCyclin and immunoprecipitation of PfPKS from Plasmodium extract followed by kinase assay of PfORCl In vitro kinase experiments results indicate that among the pfcdk like kinases tested here, PfPKS in combination with RINGO shows better phosphorylation on pforcin substrate compared to PfPK6 and pfcrk4. it is clear that PfPKS is not activated in vitro in the presence of pfcyclins. Since RINGO is not an activator from Plasmodium origin, we are interested to know whether an active form of PfPKS is possible to get from P. jalciparum extract. In order to get an active form of PfPKS, we performed pull down experiments using either Nickel beads bound PfPKS following incubation with P. jalciparum extract or by immunoprecipitating PfPKS from directly from P. jalciparum extract followed by in vitro kinase assay using pforc IN or Histone HI as substrate

84 Cyclin 1 Cyclin 2 Cyclin 3 Cyclin 4 A. on bead on bead B. on bead on bead T S T S Plasmodium extract T T * Coomassie 32.p Histone HI c. PfORC1N PfORC1- L,.N2.,. I Cyclin 1 on bead T Cyclin 2 Cyclin 1 Cyclin 2 on bead on bead on bead S T S T S T S D. PfORC1N Plasmodium extract Cyclin 3 on bead T s Cyclin 4 on bead T s C i Autorad Coomassie Figure 48: Pull-down assay from Plasmodium extract. (A) GST-PfCyc I and GST-PfCyc2 were purified under native conditions and bound onto Glutathione beads. Bound beads were incubated with ~ 150 mg parasite extract from Troph (T) or Schizont (S) stage followed by kinase assay using Histone HI as a substrate. (B) MBP-PfCyc3 and GST-PfCyc4 were bound onto beads and incubated with parasite extract followed by kinase assay using Histone HI (1 mg) as a substrate. (C) PfCyc I and PfCyc2 were incubated with stage specific Plasmodium extract as described in Materials and methods. Kinase assay was performed using recombinant PfDRCIN (lane 1-4) and PfDRCI- L,.N2 (lane 5-8) as a substrate. (*) shows the faint band ofpforcin. 'T' stands for trophozoite and's' stands for schizont stage parasites. (0)PfCyc3 and PfCyc4 were purified and bound onto beads and incubated with parasite extract from troph (T) and schizont (S) stage. Kinase assay was performed using PfDRCIN as a substrate. In lane 5, alone PfDRCIN was taken as a control. In (A), (B), (C), and (0) upper panel shows the autoradiogram and lower panel shows the coomassie stained gel to show the loading control.

85 Results: Chapter IV For pull-down experiments, Ni2+ beads containing His6- tagged PfPK5 protein was incubated with P. jalciparum extract (late Troph/ early schizont stage) followed by kinase reaction using either wild type pforcin or pforcl~n2. The results indicate that PfPK5 IS autophosphorylated In all the cases and Histone HI IS also phosphorylated suggesting the activation of PfPK5 following incubation with Plasmodium extract. Wild type pforcin but not PfORCl~N2 was phosphorylated under the same experimental conditions suggesting the specificity of pforcin phosphorylation using activated PfPK5. The coomassie stained gel shows the equivalent loading of all the proteins (Figure 49). Further, we performed immunoprecipitation of PfPK5 from the Plasmodium extract using anti-chicken PfPK5 antibody as described in Materials and methods. After immunoprecipitation, we performed the kinase assay using either Histone HI or GST-pfORCl as substrate. The results show that the control Histone HI protein phosphorylated by immunoprecipitated PfPK5 from the Plasmodium extract. Wild type pforcin but not the pforcl~n2 is also phosphorylated under the same experimental conditions confirming the specificity of phosphorylation of pforcin by PfPK5 (Figure 50). Finally, roscovitine, a specific inhibitor for CDK like kinases was shown to inhibit the phosphorylation of pforcin by immunoprecipitated PfPK5 at a concentration of 100 pm further suggesting the specificity of pforcin phosphorylation by PfPK5 (Figure 50). In summary, we have screened three CDK like kinases from Plasmodium in combination with four Plasmodium Cyclins using Histone HI (as a positive control) and pforcin as substrates in kinase assays in vitro. We find that both PfPK5 and pfcrk5 shows different affinity towards

86 PfCyclin pull down Autorad * Coomassie Figure 49: Pull down from Plasmodium extract. His-PfPK5 was purified and bound onto Ni-NTA bead and then incubated with Plasmodium extact at 4 0 C for 1 hr followed by kinase reaction using Histone HI (lane 1), PfDRCIN (lane 2) or PfDRC16 N2 (lane 3). Lane 4 contains the PfDRCl, kinase buffer and y_32p ATP. Upper panel shows the autoradiogram and bottom panel shows the coomassie stained gel. (*) shows the position ofpfdrcin.

87 PfPK51P * Autorad * Coomassie Figure 50: Immunoprecipitation from parasite extract using anti-ptpk5 antibody followed by kinase assay of PfORCl. Anti-Chicken PfPK5 antibody was incubated with Plasmodium extract (trophlschizont stage) as described in Materials and methods. Kinase assay was perfonned using (lane 1): Histone HI;. (lane 2): PfDRCIN; (lane 3): PfDRCl- ~N2 as a substrate; (lane 4) roscovitine along with PfDRCIN in the kinase reaction; (lane 5): shows the control lane containing Histone HI in kinase buffer with y)2p AIP. Bottom panel shows the coomassie stained gel. (*) shows the position of PfDRC 1 N.

88 Results: Chapter IV different Cyclins (PfCyc-2 and PfCyc-4 for PiPK6 and PfCyc-1 and PfCyc-4 for PfCRKS) to form active kinase leading to the phosphorylation of control Histone HI. Although PiPK6 and PfCyclin combinations phosphorylate PfORC1N to some extent, no phosphorylation of PfORC1 is observed using PfCRKS and PfCyclin combinations suggesting different substrate specificity of these different kinases. Interestingly, PiPKS is not active in combination with any of the Cyclins. However RINGO, a positive stimulator for mammalian CDKS, activated PiPKS that can phosphorylate both Histone HI and wild type PfORC1N strongly but not a deletion form of PfORC 1N lacking the putative CDK phosphorylation sites. In consistent with the in vitro kinase assay using recombinant proteins, pull-down experiments using PfCyclins or PiPKS and Plasmodium extract also confirm the specificity of PfORC1N phosphorylation. Further, phosphorylation of PfORC1N by imrnunoprecipitated PiPKS was further inhibited by roscovitine, an inhibitor for CDK related kinases. These results clearly suggest that PfORC 1 may serve as a specific substrate for PiPKS kinase activity during DNA replication and cell-cycle regulation in Plasmodium. The results of the kinase assay are summarized in table

89 Results: Chapter IV Table 10: In vitro kinase and cyclin screening A. Activity with HistoneHI Kinase Cyelin Substrate PfCyc-l PfCyc-2 1. PfPK PfCRK5 + - PfCyc-3 PfCyc-4 RINGO HistoneHI HistoneHI B. Activity with PfORCIN Kinase Cyclin Substrate PfCyc-l PfCyc-2 1. PfPK PfPK PfCRK5 - - PfCyc-3 PfCyc-4 RINGO PfDRCIN - + ND PfDRCIN - - ND PfDRCIN In vivo kinase and cyclin screening A. PfKinase pull down from Plasmodium Jalciparum extract Substrate Cyclin PfCyclinl PfCyclin2 PfCyclin3 PfCyclin4 HistoneHI PfDRCIN B. PfCyclin pull down from Plasmodium Jalciparum extract Substrate Kinase PfPKS HistoneHI +++ PfDRCIN ++ PfDRCILlli Strong phosphorylation activity Moderate phosphorylation activity Weak phosphorylation activity ND No activity Not done

90 Chapter V Formation of PfORe1 replication foci during different developmental stages in Plasmodium falciparum

91 Results: Chapter V 5.1 Co-localization of PfORC1 and PfPCNA in different developmental stages of the parasite In eukaryotes DNA replication takes place in nuclear foci termed as replication foci. These foci represent the replication factories which contain several components involved directly or indirectly in DNA replication. Formation of replication foci occurs at the time of DNA synthesis. These replication foci are either static in nature with DNA forks passing through them or they are dynamic with varying distribution within nuclei during cell cycle process. Within one nucleus replication foci assemble and disassemble during the S-phase (Leonhardt et al., 2000). Origin Recognition Complex serves as a landing pad for other replication factors at the site of origin DNA to initiate replication. Thus replication foci should coincide with the origin of DNA replication which contains ORC subunits along with the other factors to start replication. PCNA is a marker for replication factories and it is highly conserved from yeast to humans. It is a cofactor of DNA polymerase. PCNA is loaded onto the DNA by replication factor-c (RF-C) in an ATP dependent manner. PCNA then serves as a sliding clamp for the processive DNA pol 5 and pol. It is also considered as an essential DNA replication factor. PCNA is a multifunctional protein which interacts with several proteins during the cell cycle. PCNA-interacting proteins contain a conserved motif termed as PIP-box (PCNA-interaction protein box). However, several proteins contain the conventional "PIP-box" and interact with the interdomain connecting loop of PCNA through a conserved sequence of eight aminoacids [QxxMjLjIxxFF jfy] but the functional relevance of these interactions is established only for few proteins (Senga et al., 2006). In P. Jalciparum PCNA is expressed during early trophozoite stage of the parasite where DNA replication also initiates during IDC (Kilbey et al,

92 Results: Chapter V 1993). Consequently its expression pattern coincides with replication window of the Plasmodium. In order to find out whether PfORC 1 is a component of replication foci at the onset of DNA replication initiation in P. jalciparum, we performed colocalization studies of PfORC 1 and PfPCNA in different developmental stages of the parasite life cycle. Immuno-colocalization experiments show the presence of PfPCNA during ring stage parasites where PfORC 1 is not present at all. With further parasite development, during early trophozoite stage, both PfPCNA and PfORC1 are visible with a diffused staining pattern. As the parasites mature further, these parasites show very distinct spots that mostly co-localize with each other. We believe that these foci are DNA replication foci since they contain PfPCNA, the marker for DNA replication. These foci slowly separate from each other until late schizont stage where ORC1 foci are completely diminished whereas PfPCNA foci are still found intact suggesting degradation of PfORC 1 during late schizont stage. The number of foci increases till late trophozoite/ early schizont stages following which these numbers decrease (Figure 51). The disappearance of PfORC1 at the late schizont stage was further confirmed by western blot analysis using PfORC1 polyclonal antibodies against parasite lysate from trophozoite stage and late schizont stage. Western blot analysis shows the presence of PfORC1 band during trophozoite stage that is absent during late schizont stage confirming the results of immunofluorescence analysis. Interestingly, PfPCNA is present in both the stages suggesting the regulation of PfORC 1 but not PfPCNA during parasite development. The coomassie stained membrane following transfer shown equivalent amount of protein loaded in each lane (Figure 52)

93 DAPI PfORC1 Pf PCNA Merged 1 Merged 2 Phase Ring Early Troph Mid- Troph Late- Troph Early Schizont Mid- Schizont Late Schizont Figure 51: Co-localization of PfORCI and PfPCNA in different asexual stages of P. Jalciparum. Glass slides containing thin smears of different asexual stages of P. Jalciparum were made. Indirect immunoflu orescence assay was performed with affinity purified PfORCl (anti-rabbit) and PfPCNA (anti-mouse) antibodies as described under Materials and methods. PCNA, a replication marker, was present from late ring/ early troph. stage to late schizont stage while PfORCl was absent in late schizont stage. From mid-trophozoite to late trophozoite stage, PfORCl and PfPCNA co-localized showing the presence of active replication sites.

94 (1- PfORC III ql' Coomassie.~ (1- PfPCNA Figure 52: Western blot analysis of parasite lysate obtained from trophozoite and late schizont stage using a-pforcl and a-pcna antibodies. Whole cell extract from trophozoite and late schizont stage parasites were boiled with 2XLlemmeli buffer. Samples were resolved on 8% SDS-PAGE and transferred onto PVDF membrane followed by western blot using respective antibodies. Coomassie stained gel is shown as a loading control.

95 Results: Chapter V Replication foci dynamics in P. falciparum and correlation of these foci with DNA replication pattern in the parasites In mammalian cells, replication foci appear at the site of DNA replication during S-phase. The pattern of replication foci is often co-localized with BrdU incorporation suggesting these are the sites where active replication takes place. The number of replication foci varies considerably among different species. In mammalian cells during early S phase the number of active replication sites is ~ 126 but as the stage progress towards mid-to-iate S-phase the number of these foci decreases (Berezney et al., 2000). In order to investigate the replication foci dynamics in developmental stages of P. /alciparum, we observed the foci pattern of PfORC1 and PfPCNA in co-localization experiment. We have shown the co-localization of pforc 1 and PfPCNA in different asexual stages where majority of pforc1 and PfPCNA foci merged with each other during trophozoite stage. As the stage progresses, these foci start detaching from each other during late trophozoite to mid schizont stage. In the late schizont stage pforc1 expression is almost diminished but PfPCNA foci are found intact in the parasite, although the numbers of PCNA foci are reduced. In order to find out whether these foci correlate with the replication timing during asexual stage of the parasite life-cycle, we calculated the total number of foci versus merged foci of pforc1 and PfPCNA in each stage. These values were plotted graphically and were compared with the pattern of DNA replication during asexual stage of the parasite. When we plot number of pforc 1 and PfPCNA foci during different developmental stages, we find DNA replication in the parasite starts around ~22-24 hrs post erythrocytic invasion and continues till schizont stage with majority of DNA synthesis occurring during hrs. Based

96 Results: Chapter V on the various published results on DNA replication timing in the parasites, we obtained a general pattern of DNA replication during different developmental stages as shown in Figure 53A. PfORC 1 and PWCNA foci formation starts in early trophozoite stage and as the stage progresses the number of foci also increases till late trophozoite stage. Subsequently, the number of foci does not change significantly. In the late schizont stage, number of pforc 1 foci almost diminishes and the same for PWCNA decreases too (Figure 53B). Maximum number of pforc 1 and PWCNA foci merged with each other during mid-to late trophozoite stage (Figure 53C). This pattern was nicely correlated with the pattern of DNA replication where maximum amount of replication takes place during mid-to late trophozoite stage (Figure 53A) Replication foci formation in PfORCI-GFP cell line To investigate the intracellular localization of pforc 1 in live P. Jalciprum infected blood cells, the N-terminus of pforc1 ORF (1-238 aa) was cloned into parl vector between Acc65I and Avril restriction sites where GFP was located at C-terminus of the fusion construct. The coding sequence of the pforc1n terminus contains the putative nuclear localization signal as well as the two Cyclinj CDK phosphorylation sites. GFP-fusion construct was transfected into the parasite infected RBC at early ring stage as described in materials and methods. The parasites were selected against drug (WR99210) for few weeks and stable transfected parasite line expressing pforc1n-gfp was obtained. Figure 54 shows the localization of pforc1-gfp in different stages of the parasite life-cycle. We find punctate foci of pforc1 in trophozoite and schizont stages. This result is similar to the punctate pattern of pforc 1 in fixed parasite cells following immunofluorescence experiments using pforc1 antibodies. The expression of pforc1-gfp in parasite cells was

97 A. B..!!? I/) CI).r:. -c: :>. I/) <C 6 0 Z C 40 -c: CI) Co> 20 ~ CI) a NNN N C'?..,..,.., ~ ~... N CDO"'ll:l"CDCOON"'II:I" CDO"'ll:l"CO ~ ~ Time in hours (post invasion) RING TROPH. SCHIZONT Foci count 25 S 20 "iii!'" 15 "u.2 10 '0 ci c: 5 0 ET MT LT/ES MS LS C. Merged foci 30 S 25 "iii ~ 20.. a. ~ 15 2 '0 10 ci c: 5 0 ET MT LT/ES MS LS Figure 53: Replication foci dynamics in PJalciparum. (A) Graph shows the hypoxanthine uptake assay at different time points of the parasite growth where maximum DNA synthesis takes place at hr post invasion. (B) Total no ofpforci and PfPCNA foci during different developmental stages of the parasite life-cycle. (C) No. of merged foci of PfORCI and PfPCNA in erythrocyte developmental cycle of P Ja lciparum representing the replication factories. - e,",

98 DAPI with Cell PfORC1-GFP Merged Early troph Mature troph Mid schizont Mid-to-Iate schizont Figure 54: Localization of PfORCI-GFP in P. Jalciparum. Localization of PfORC l -GFP in stably transfected parasite line was monitered in dufferent stages of the parasite ( as mentioned at the right) by confocal microscopy. DAPI shows the nuclear staining within the parasite.

99 Results: Chapter V further confirmed by western blot analysis using anti-gfp antibody (Figure 55). The presence of PfORC1N-GFP spots during late schizont stage can be attributed to the absence of C-terminal region that can be responsible for the degradation of ORC 1 at the late stage. 5.2 Perturbation of DNA replication by Hydroxyurea and its effect on PfORCl foci formation The time of initiation of DNA synthesis in P. Jalciparum has been determined by pulse-labeling methods using radioactive [3H] hypoxanthine uptake where it has been found that replication starts at early trophozoite stage (~22-24 hour post infection) and continues till mid to late schizont stage (Inselburg and Banyal, 1984). Although the majority of DNA synthesis takes place during mid trophozoite to late schizont stage in P. Jalciparum, different replication inhibitors have also been used to study their effect on DNA replication in different Plasmodium species. Hudroxyurea is a cell cycle specific S-phase inhibitor. It acts as an inhibitor of ribonucleotide reductase and blocks DNA synthesis by preventing the dntp pool expansion that normally occurs at G 1 / S phase. To evaluate the effect of replication inhibitors on DNA synthesis and further on replication foci formation, we used replication inhibitor hydroxyurea in P. Jalciparum blood stage culture and followed its effect on pforc 1 /pfpcna foci formation. Hydroxyurea was added in the asexual synchronized parasites at the early ring stage (~7 hr post infection) at the concentration of 100 pm/ml. The effect of hydroxyurea (HU) on parasite growth and development was examined at regular time intervals by Giemsa staining of parasite infected blood smears. In the HU-treated samples, parasites were arrested in late ring/early

100 GFP breakdown Figure 55: Expression ofpforcl-gfp in P. Jalciparum was examined by western blot analysis using anti-gfp antibody. (*) shows the position ofpfdrci-gfp.

101 Results: Chapter V trophozoite stage that corresponded to the time of initiation of DNA synthesis. Under the same experimental conditions, the HU-untreated parasites preceded through normal development stages (Figure 56). To further check the status of replication initiation proteins in hydroxyurea treated parasites, we performed co-localization study with anti-pforc 1 (rabbit) and with anti-pcna (mice) antibodies. Immunofluorescence assay results indicate that untreated parasites follow normal growth progression with ORC 1 and PCNA foci formation during trophozoite stage (Figure 57). While PiPCNA foci are still visible during late schizont stage, PfORCl foci are completely absent during the same stage suggesting degradation of PfORCl at the late schizont stage as shown earlier (Figure19). Interestingly, hydroxyurea treated samples show severe growth defect following the addition of drug at early ring stage. Most parasites were stuck at the late ring/ early trophozoite stage with a diffused staining pattern for both PfORC 1 and PiPCNA. The intensity of both PiPCNA and PfORCl signal is reduced significantly and the nuclei are seemed to be fragmented following ~40 hours of incubation of the parasites in the presence of hydroxyurea suggesting that the addition of hydroxyurea blocks replication foci formation and DNA replication leading to the overall growth defect of the parasites. These results also indicate that PfORCl foci formation is essential for DNA replication initiation in the parasites (Figure 57)

102 Post treatment (time) o hr + 32 hr -HU + HU Figure 56: Giemsa staining of P. falciparum with or without hydroxyurea (HU) treatment. Synchronized parasites were treated with 100 ~ HU at ring stage. Samples were collected at different time points from HU untreated and treated parasites. Giemsa staining was performed to check the effect of drug on parasite growth.

103 Time DAPI PfORC1 PCNA Merged1 Merged 2 Cell (post treatment) ~ , Ring - 0 hr Troph - 30 hr -HU Schizont - 46 hr Ring Troph - 0 hr - 30 hr + HU Schizont - 40 hr Figure 57 : Effect of Hydroxyurea on parasite growth and replication. Early ring stage parasites were treated with Hydroxyurea at the concentration of 100 ~/m l and samples were taken out at regular time interval for immunuofluorescence analysis. Thin smears were made on glass slides with HU-untreated and HU-treated samples and IFA was done using PfORCIC and PtpCNA antibodies as described in Materials and methods. 'Merged l' shows the co-localization of PfORCl and PtpCNA with nuclear stain DAPI while 'Merged 2' shows the co-localization of PfORCl and PtpCNA antibodies.

104 Results: Chapter V 5.3 Stabilization of PfORCl by proteasomal inhibitor MG132 The largest subunit of ORC, ORC 1, is differentially regulated in higher eukaryotes during cell cycle. In rapidly proliferating mammalian cells, human ORC 1 is expressed and targeted to chromatin as cells exit mitosis and pre-replicative complexes are formed. As the cells enter S phase, human ORC 1 is ubiquitinated and degraded by proteasome mediated pathways (Mendez et al., 2002). In Chinese hamster ovarian cells ORC1 is selectively mono-and di-ubiquitinated and it is removed from chromatin after the cells enter S phase (Li and DePamphilis, 2002). In contrast to human ORC1, ScORC1 is attached to the chromatin throughout the cell-cycle. It is interesting to note that PfORC1 is also regulated through developmental stages in Plasmodium. Both immunofluorescence and western blot data indicate that PfORC 1 is visible during late ring/early trophozoite stage just before DNA replication initiation in the parasites. The protein stays in the parasites till late schizont stage where it is selectively degraded. The selective degradation of PfORC 1 may ensure the regulation of DNA replication in the parasites. In order to find out whether PfORC1 is degraded by proteasomal mediated pathways during late schizont stage, we used MG 132, a proteasomal inhibitor. The drug was added into the in vitro parasite culture at -40 hours and followed for further -8 hours to see the effect of drug on parasite growth, ORC1 foci and ORC1 protein stability. Initially we used different concentrations (100 nm, 1 pm and 10 pm, respectively) of inhibitor to ascertain the effect of drug on parasite growth. Giemsa staining of glass slides containing parasites from different stages following drug treatment at different concentration reveals that parasite

105 Results: Chapter V growth is stalled at 1 pm and 10 pm MG 132 concentration whereas 100nM drug concentration shows milder effect on parasite growth when compared to the untreated parasites that shows mature schizont formation at ~48 hrs (~8 hrs following drug treatment) (Figure 58). Therefore, we decided to use 100 nm MG132 concentrations to check the effect of MG132 on ORC1 foci formation and protein stability. As a control100nm DMSO solution was also used to rule out the possibility of any effect of solvent on the parasite growth. Immunofluorescence analysis was performed to check the fate of PfORC 1 foci in MG 132-treated (100 nm) and untreated samples at different time points. MG132 untreated samples show PfORC1 expression at '0 hr' ( ~ 40 hr post invasion followed by absence of PfORC1 expression at ~8 hr (~48 hr post invasion) as shown previously (Figure 59). Interestingly, MG 132 treated samples show stabilization of PfORC1 foci at the late schizont stage (-8 hr) under the same experimental conditions. These results suggest that MG 132 inhibits proteasomal mediated degradation of PfORC 1 at the late schizont stage leading to the re-establishment of PfORC1 foci at late schizont stage. To further confirm the result of IF A, we performed western blot analysis to check the protein level in MG312-treated and untreated samples. Samples were boiled with 2XLamellie buffer and loaded onto 8% SDS PAGE followed by western blot analysis using anti-pforc1 antibodies or anti-pfpcna antibodies. In untreated samples, PfORC1 band was visible at '0' time point (at the time of drug addition) whereas the same band was not found at the late schizont stage (-8 hr time point) (Figure 60). In contrast, the MG 132 treated samples showed the presence of PfORC1 band at - 48hr time point. It is interesting to note that the ORC 1 band in MG 132 treated sample is shifted upward suggesting post translational modification of PfORC 1 at the late stage. However, PCNA was present in

106 Time (hrs) MG132 o 100 nm Figure 58: Giemsa staining of MG132 treated and untreated samples. Synchronized parasites were treated with different concentrations of MG 132, as indicated on the right. Samples were taken out at different time points (as indicated on top) to check the morphological changes by Giemsa staining.

107 DAPI PfORC1 MERGED CELL Lfl ES (- 40 hr) LS (- 48 hr) - MG1 32 LS (- 48 hr) + MG132 Figure 59: Indirect IF A of MG 132 treated and untreated parasites. Synchronized parasites were treated with 100nM MG132 at - 40 hour post infection and followed till late schizont stage (-48 hrs). IFA was performed with anti PfORCl C (1 :4000) antibodies as described in Materials and methods. Slides were scanned under fluorescent NIKON microscope.