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• W1997091164 abstract "DNA replication in Plasmodium parasites takes place at multiple distinct points during their complex life cycle in the mosquito and vertebrate hosts. Although several parasite proteins involved in DNA replication have been described, the various mechanisms engaged in DNA metabolism of this major pathogen remain largely unexplored. As a step toward understanding this complex network, we describe the identification of Plasmodium falciparum replication protein A large subunit (pfRPA1) through affinity purification and mass spectral analysis of a purified 55-kDa factor. Gel retardation experiments revealed that pfRPA is the major single-stranded DNA binding activity in parasite protein extracts. The activity was expressed in a cell cycle-dependent manner with peak activities in late trophozoites and schizonts, thus correlating with the beginning of chromosomal DNA replication. Accordingly, the pfrpa1 message was detected in parasites 20–24 h post-invasion which is in agreement with the expression of other P. falciparumDNA replication genes. Our results show that pfRPA1 is encoded by an unusual 6.5-kb transcript containing a single open reading frame of which only the C-terminal 42% of the deduced protein sequence shows homologies to other reported RPA1s. Like the orthologues of other protozoan parasites, pfRPA1 lacks the N-terminal protein interaction domain and is thus remarkably smaller than the RPA1s of higher eukaryotes. DNA replication in Plasmodium parasites takes place at multiple distinct points during their complex life cycle in the mosquito and vertebrate hosts. Although several parasite proteins involved in DNA replication have been described, the various mechanisms engaged in DNA metabolism of this major pathogen remain largely unexplored. As a step toward understanding this complex network, we describe the identification of Plasmodium falciparum replication protein A large subunit (pfRPA1) through affinity purification and mass spectral analysis of a purified 55-kDa factor. Gel retardation experiments revealed that pfRPA is the major single-stranded DNA binding activity in parasite protein extracts. The activity was expressed in a cell cycle-dependent manner with peak activities in late trophozoites and schizonts, thus correlating with the beginning of chromosomal DNA replication. Accordingly, the pfrpa1 message was detected in parasites 20–24 h post-invasion which is in agreement with the expression of other P. falciparumDNA replication genes. Our results show that pfRPA1 is encoded by an unusual 6.5-kb transcript containing a single open reading frame of which only the C-terminal 42% of the deduced protein sequence shows homologies to other reported RPA1s. Like the orthologues of other protozoan parasites, pfRPA1 lacks the N-terminal protein interaction domain and is thus remarkably smaller than the RPA1s of higher eukaryotes. Plasmodium falciparum causes one of the most life-threatening parasitic diseases in humans being responsible for up to 2 million deaths per year. Malaria pathogenesis is associated with the intracellular erythrocytic stage of the life cycle of the parasite involving repeated rounds of invasion, growth, and schizogony. Parasites that eventually differentiate into gametocytes are taken up by the female anopheline vector where zygote formation and sporogony take place. Sporozoites injected into a human host by the bite of an infective mosquito invade hepatocytes and, after schizogony, release thousands of merozoites capable of invading red blood cells. During each replication event, the timing, rate, and extent of genome multiplication have to be controlled appropriately and coordinated at each developmental stage. Most of the studies on DNA replication in P. falciparum have been conducted during the erythrocytic stages. Several genes involved in eukaryotic chromosomal DNA replication and their encoded proteins have been identified in this parasite, including DNA polymerases α (DNA pol α) 1The abbreviations used are: DNA pol α and δ, DNA polymerases α and δ; ssDNA, single-stranded DNA; RPA, replication protein A; RPA1, large subunit of RPA; EMSA, electromobility shift assay; hpi, h post-invasion; dsDNA, double-stranded DNA; PMSF, phenylmethylsulfonyl fluoride; ORF, open reading frame; DTT, dithiothreitol; aa, amino acid; oligo, oligonucleotides; MS, mass spectroscopy. (1Chavalitshewinkoon P. de Vries E. Stam J.G. Franssen F.F. van der Vliet P.C. Overdulve J.P. Mol. Biochem. Parasitol. 1993; 61: 243-253Crossref PubMed Scopus (21) Google Scholar, 2White J.H. Kilbey B.J. de Vries E. Goman M. Alano P. Cheesman S. McAleese S. Ridley R.G. Nucleic Acids Res. 1993; 21: 3643-3646Crossref PubMed Scopus (34) Google Scholar) and δ (DNA pol δ) (3Fox B.A. Bzik D.J. Mol. Biochem. Parasitol. 1991; 49: 289-296Crossref PubMed Scopus (19) Google Scholar, 4Ridley R.G. White J.H. McAleese S.M. Goman M. Alano P. de Vries E. Kilbey B.J. Nucleic Acids Res. 1991; 19: 6731-6736Crossref PubMed Scopus (54) Google Scholar), proliferating cell nuclear antigen (5Kilbey B.J. Fraser I. McAleese S. Goman M. Ridley R.G. Nucleic Acids Res. 1993; 21: 239-243Crossref PubMed Scopus (40) Google Scholar), and topoisomerases I (6Tosh K. Kilbey B. Gene (Amst.). 1995; 163: 151-154Crossref PubMed Scopus (27) Google Scholar) and II (7Cheesman S. McAleese S. Goman M. Johnson D. Horrocks P. Ridley R.G. Kilbey B.J. Nucleic Acids Res. 1994; 22: 2547-2551Crossref PubMed Scopus (38) Google Scholar). It has been shown that expression of these genes follows a stage-specific pattern coinciding with the beginning of chromosomal replication which starts 28–31 h after merozoite invasion and continues through most of schizogony (8Inselburg J. Banyal H.S. Mol. Biochem. Parasitol. 1984; 10: 79-87Crossref PubMed Scopus (87) Google Scholar). Due to their complex life cycle and constant immunological pressure exerted by their hosts, the processes of DNA metabolism in Plasmodium parasites must be both very efficient and flexible. The high degree of genetic variability observed in this parasite (9Scaife J.G. Genet. Eng. 1988; 7: 57-90Google Scholar, 10Weber J.L. Gene (Amst.). 1987; 52: 103-109Crossref PubMed Scopus (117) Google Scholar) and the fact that DNA replication occurs at five distinct developmental points, namely intrahepatocytic schizogony, intraerythrocytic schizogony, microgametogenesis, premeiotic DNA synthesis, and sporozoite development (11White J.H. Kilbey B.J. Parasitol. Today. 1996; 12: 151-155Abstract Full Text PDF PubMed Scopus (32) Google Scholar) indicate the operation of highly regulated mechanisms of DNA replication, recombination, and repair. In fact, dynamic processes of DNA metabolism may be one reason for the outstanding success of this parasite because this supports rapid adaptation to environmental challenges such as immune pressure and action of antimalarial drugs. In addition, the unusually high AT content of ∼80% in the P. falciparum genome (12McCutchan T.F. Dame J.B. Miller L.H. Barnwell J. Science. 1984; 225: 808-811Crossref PubMed Scopus (133) Google Scholar, 13Pollack Y. Katzen A.L. Spira D.T. Golenser J. Nucleic Acids Res. 1982; 10: 539-546Crossref PubMed Scopus (110) Google Scholar) may also indicate peculiarities in the replication machinery of the parasite. Hence, investigation of the components involved in DNA metabolism of P. falciparum might reveal features unique to this parasite and may consequently lead to the identification of new potential drug targets for malaria therapy. The eukaryotic single-stranded (ss) DNA-binding protein replication protein A (RPA) plays essential roles in various aspects of DNA metabolism, including replication, recombination, and repair (for review see Ref. 14Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1188) Google Scholar). The protein has high affinity for ssDNA (15Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (295) Google Scholar, 16Wobbe C.R. Weissbach L. Borowiec J.A. Dean F.B. Murakami Y. Bullock P. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1834-1838Crossref PubMed Scopus (259) Google Scholar, 17Wold M.S. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (369) Google Scholar) and binds with much lower affinity to double-stranded DNA (dsDNA) and RNA (18Kim C. Snyder R.O. Wold M.S. Mol. Cell. Biol. 1992; 12: 3050-3059Crossref PubMed Scopus (242) Google Scholar, 19Wold M.S. Weinberg D.H. Virshup D.M. Li J.J. Kelly T.J. J. Biol. Chem. 1989; 264: 2801-2809Abstract Full Text PDF PubMed Google Scholar). RPA was originally identified as a factor being absolutely required for SV40 replication in vitro (15Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (295) Google Scholar, 16Wobbe C.R. Weissbach L. Borowiec J.A. Dean F.B. Murakami Y. Bullock P. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1834-1838Crossref PubMed Scopus (259) Google Scholar, 17Wold M.S. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (369) Google Scholar). In this system RPA interacts with large T-antigen and DNA pol α/primase, and these interactions seem to be important in loading DNA pol α/primase onto the RPA-coated unwound origin of replication to allow initiation of DNA synthesis to occur (20Brown G.W. Melendy T.E. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10227-10231Crossref PubMed Scopus (37) Google Scholar, 21Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (285) Google Scholar, 22Melendy T. Stillman B. J. Biol. Chem. 1993; 268: 3389-3395Abstract Full Text PDF PubMed Google Scholar). Furthermore, RPA is involved in unwinding of dsDNA (23Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (189) Google Scholar, 24Georgaki A. Strack B. Podust V. Hubscher U. FEBS Lett. 1992; 308: 240-244Crossref PubMed Scopus (87) Google Scholar, 25Georgaki A. Hubscher U. Nucleic Acids Res. 1993; 21: 3659-3665Crossref PubMed Scopus (39) Google Scholar, 26Treuner K. Ramsperger U. Knippers R. J. Mol. Biol. 1996; 259: 104-112Crossref PubMed Scopus (79) Google Scholar), stimulation of DNA pol α/primase activity, and replication factor C- and proliferating cell nuclear antigen-dependent DNA synthesis by DNA pol δ (27Braun K.A. Lao Y., He, Z. Ingles C.J. Wold M.S. Biochemistry. 1997; 36: 8443-8454Crossref PubMed Scopus (115) Google Scholar, 28Erdile L.F. Heyer W.D. Kolodner R. Kelly T.J. J. Biol. Chem. 1991; 266: 12090-12098Abstract Full Text PDF PubMed Google Scholar, 29Kenny M.K. Lee S.H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9757-9761Crossref PubMed Scopus (165) Google Scholar, 30Tsurimoto T. Stillman B. EMBO J. 1989; 8: 3883-3889Crossref PubMed Scopus (190) Google Scholar, 31Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar, 32Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1961-1968Abstract Full Text PDF PubMed Google Scholar). RPA exists as a heterotrimeric complex consisting of subunits of ∼70, 34, and 14 kDa in all eukaryotic organisms examined (14Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1188) Google Scholar). Among these, genes coding for RPA subunits have been identified and described in two protozoan parasites. In Crithidia fasciculata the large subunit of RPA (RPA1) is only 51 kDa in size (33Brown G.W. Hines J.C. Fisher P. Ray D.S. Mol. Biochem. Parasitol. 1994; 63: 135-142Crossref PubMed Scopus (27) Google Scholar), and the predicted size of the large subunit of Cryptosporidium parvum RPA is 54 kDa (34Zhu G. Marchewka M.J. Keithly J.S. FEMS Microbiol. Lett. 1999; 176: 367-372Crossref PubMed Google Scholar). In both RPA homologues the N-terminal protein-interaction domain is lacking, which has been shown to be required for stimulation of DNA pol α/primase and important in DNA recombination and repair (35Firmenich A.A. Elias-Arnanz M. Berg P. Mol. Cell. Biol. 1995; 15: 1620-1631Crossref PubMed Google Scholar, 36Gomes X.V. Wold M.S. Biochemistry. 1996; 35: 10558-10568Crossref PubMed Scopus (90) Google Scholar, 37Kim D.K. Stigger E. Lee S.H. J. Biol. Chem. 1996; 271: 15124-15129Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Lin Y.L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 39Longhese M.P. Plevani P. Lucchini G. Mol. Cell. Biol. 1994; 14: 7884-7890Crossref PubMed Scopus (115) Google Scholar). During our studies of protein-DNA interactions in P. falciparum promoters, we observed the major ssDNA binding activity in parasite nuclear extracts with binding properties resembling those described for RPAs of other organisms. Affinity purification and mass spectrometric analysis identified a 55-kDa protein as the P. falciparum RPA large subunit homologue (pfRPA1). Interestingly, as in the apicomplexan parasite C. parvum and the trypanosomatid C. fasciculata, this protein lacks the N-terminal protein-interaction domain. Further sequence analysis revealed that the P. falciparum rpa1 transcript is unusually long (∼6.5 kb) and encodes a single exon ORF potentially coding for a predicted protein of 1145 amino acids (aa). However, the region sharing homology with other RPA large subunits consists of only the C-terminal 42%. The biological significance of this unusual organization remains unknown. We have also shown that the presence of pfrpa1 message and pfRPA1 activity correlate with timing of chromosomal DNA replication as it has been described for other P. falciparum replication factors. The identification of a key molecule involved in DNA metabolism in P. falciparum provides a deeper understanding of such essential processes in this parasite and may lead to new pharmacological intervention strategies. P. falciparum3D7/K+ parasites were cultured in 150-mm Petri dishes at 5% hematocrit as described previously (40Trager W. Jenson J.B. Nature. 1978; 273: 621-622Crossref PubMed Scopus (187) Google Scholar) in RPMI medium supplemented with 0.5% albumax (Invitrogen). Growth synchronization was achieved by sorbitol lysis (41Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2855) Google Scholar) which eliminates all but ring stage parasites. Protein extracts were prepared with modifications according to Hoppe-Seyler et al. (42Hoppe-Seyler F. Butz K. Rittmuller C. von Knebel D.M. Nucleic Acids Res. 1991; 19: 5080Crossref PubMed Scopus (64) Google Scholar). Parasites were released from red blood cells by saponin lysis and washed twice in 1× phosphate-buffered saline. The parasite pellet was resuspended in ice-cold lysis buffer (20 mmHepes, pH 7.8, 10 mm KCl, 1 mm EDTA, 1 mm DTT, 1 mm PMSF, 0.65% Nonidet P-40) and incubated for 5 min on ice. Nuclei were pelleted at 2500 ×g for 5 min, and the supernatant containing cytoplasmic proteins was removed and stored at −80 °C. The nuclear pellet was washed twice in lysis buffer before resuspension in 1 pellet volume of extraction buffer (20 mm Hepes, pH 7.8, 800 mmKCl, 1 mm EDTA, 1 mm DTT, 1 mmPMSF, 3 μm pepstatin A, 100 μm l-1-tosylamido-2-phenylethyl chloromethyl ketone, 10 μm leupeptin). After vigorous shaking at 4 °C for 30 min, the extract was cleared by centrifugation at 13,000 ×g for 30 min. The supernatant (nuclear proteins) was diluted with 1 volume of dilution buffer (20 mm Hepes, pH 7.8, 1 mm EDTA, 1 mm DTT, 30% glycerol) and stored at −80 °C. 1 liter of parasite culture (5–8% parasitaemia) yielded ∼2–4 mg of nuclear proteins and 100–200 mg of cytoplasmic proteins. Single-stranded oligonucleotides were end-labeled with [γ-32P]dATP and T4 polynucleotide kinase (Amersham Biosciences) according to the supplier's instructions. The double-stranded oligonucleotide 5B1c was obtained by incubating equimolar amounts of complementary oligonucleotides 5B1f and 5B1rc in 1× React3 (Invitrogen) at 95 °C for 5 min followed by slow cooling to room temperature in a heating block. 5B1c was either labeled with [γ-32P]dATP and T4 polynucleotide kinase (see above) or with Klenow enzyme in a fill-in reaction by incubating 1 pmol of DNA in 1× React2 buffer (Invitrogen) in the presence of 50 μm dATP/dGTP/dTTP and 10 μCi (3000 Ci/mmol) of [α-32P]dCTP at 30 °C for 20 min. Probes were purified using Sephadex G-25 spin columns (AmershamBiosciences). The sequences of probes and competitors are shown in Table I.Table IOligonucleotide probes and competitors used in EMSAsOligonucleotide nameNucleotide sequence 5′ → 3′5B1rcTTC TCT TTC TAT CTA TAT TAT CTA CCA CAT5B1fAGA AAT GTG GTA GAT AAT ATA GAT AGA AAG5B1fmut1GTG GGC GTG GTA GAT AAT ATA GAT AGA AAG5B1fmut2AGA AAT TCT TCG GAT AAT ATA GAT AGA AAG5B1fmut3AGA AAT GTG GTA TGC GGC ATA GAT AGA AAG5B1fmut4AGA AAT GTG GTA GAT AAT GCG TGC AGA AAG5B1fmut5AGA AAT GTG GTA GAT AAT ATA GAT GTG GGTMutated hexanucleotide stretches in 5B1mut1–5 are highlighted in bold. Open table in a new tab Mutated hexanucleotide stretches in 5B1mut1–5 are highlighted in bold. EMSA reactions were carried out by incubating 1–2 μg of crude nuclear proteins, 5–10 μg of crude cytoplasmic proteins, or ∼0.1–0.5 ng of purified protein (see below) with 5 fmol of radiolabeled probe in EMSA buffer (20 mm Hepes, pH 7.8, 60 mm KCl, 0.5 mm EDTA, 2 mm DTT, 2 mmMgCl2, 25 mm ZnCl2, 0.1% Triton X-100, 10% glycerol) containing 100–500 ng of poly(dI-dC) as nonspecific competitor DNA in a 20-μl reaction volume for 20 min at room temperature. Binding reactions were analyzed on a 6% polyacrylamide gel in 0.5% TBE. For competition experiments the labeled probe was added 10 min after incubation of protein and competitor DNA. In EMSAs using purified proteins poly(dI-dC) was omitted, and bovine serum albumin (10 μg) was added. To assess the molecular weight of the ssDNA-protein complex ∼4 or 20 μg of crude nuclear or cytosolic protein extracts, respectively, were incubated with 60 fmol of labeled 5B1f oligo in EMSA buffer as described, followed by exposure to UV light for 10 min in a Stratalinker 1800 (Stratagene). Protein-DNA complexes were incubated at 95 °C in 1× SDS sample buffer for 5 min and separated on a 12% SDS-polyacrylamide gel. Gels were dried and analyzed by autoradiography. Prestained molecular weight markers were used to estimate the molecular mass of the ssDNA-protein complexes. ∼6–10 or 20–40 μg of crude nuclear or cytosolic protein extracts, respectively, were incubated with 20 fmol of [γ-32P]dATP-labeled 5B1f oligonucleotide (in the presence of 1.25 mm PMSF) and UV cross-linked as described above. DNA-protein complexes were incubated with 2 μg of porcine pancreas trypsin at room temperature in the same buffer. Aliquots were removed at time points indicated in Fig. 4. Reactions were stopped by the addition of SDS-PAGE sample buffer followed by incubation at 95 °C for 5 min. Samples were analyzed SDS-PAGE. Gels were dried and analyzed by autoradiography. As matrix for affinity purification, the biotinylated 90-base oligo 5B1Af (a trimer of 5B1f) was tethered to magnetic streptavidin-coated Dynabeads (Dynal) according to the supplier's instructions. Cytoplasmic extracts from a total of 7.5 liters of asynchronous parasite culture (5–8% parasitaemia) were spun at 3000 × g for 10 min to pellet cellular debris. The supernatant was incubated with 2 mg of Dynabeads (80 pmol oligo/mg beads) in binding buffer BB (20 mm Hepes, pH 7.8, 120 mm KCl, 1 mm EDTA, 1 mm DTT, 1 mm PMSF, 0.1% Triton X-100, 0.65% Nonidet P-40) on a rotating wheel for 1 h at room temperature. Dynabeads were collected by use of a magnetic stand and washed 2 times with 1 ml of wash buffer W1 (20 mm Hepes, pH 7.8, 150 mmKCl, 1 mm EDTA, 1 mm DTT, 1 mmPMSF, 0.01% Triton X-100, 5% glycerol) and 2 times with buffer W2 containing 200 mm KCl. Bound proteins were eluted stepwise twice in 200 μl of elution buffer EB1 and twice in EB2 (20 mm Hepes, pH 7.8, 2.5 mm EDTA, 1 mmPMSF, 10% glycerol) containing 0.5 and 1 m KCl, respectively. Purification from nuclear extracts was performed by incubation of 4.5 ml of crude nuclear extract (1–2 μg/μl) from a total of 2.8 liters of asynchronous parasite culture (5–8% parasitaemia) with 1 mg of Dynabeads (10 pmol of oligo/mg of beads) in binding buffer BB (20 ng poly(dI-dC)/μl) as described above. Dynabeads were washed 2 times in 300 μl of buffer W1 and once in buffer W3 (250 mm KCl). Bound proteins were eluted stepwise twice in 50 μl of EB1 followed by elution in 50 μl of EB2. Wash fractions and eluates were analyzed by EMSA and SDS-PAGE and silver staining. Samples were stored at −80 °C. A gel piece containing the Coomassie Blue-stained purified cytoplasmic ssDNA-binding factor was washed five times for 1 min each in 30 μl of 40% n-propyl alcohol followed by five 1-min washes each in 30 μl of 0.2 m NH4HCO3(50% acetonitrile). The gel piece was dried in a SpeedVac concentrator and then digested with 0.5 μg of sequencing grade modified trypsin (Promega) in 10 μl of 0.1 mNH4HCO3 for 2 h at 37 °C. The gel piece was extracted with 15 μl of 0.1% trifluoroacetic acid for 5 min followed by 15 μl of acetonitrile for 1 min. Extraction was repeated twice, and the pooled supernatants were dried in a SpeedVac concentrator. Peptides were redissolved in 10 μl of 0.1% trifluoroacetic acid, and 5 μl were used for mass spectral analysis. Separation of peptides was done on 100-μm inner diameter capillary columns packed with POROS R2 material. Mass spectral data were acquired on a TSQ7000 triple quadrupole instrument (Finnigan) with data-controlled switching between precursor ions and daughter ions (43Stahl D.C. Swiderek K.M. Davis M.T. Lee T.D. J. Am. Soc. Mass Spectrom. 1996; 7: 532-540Crossref PubMed Scopus (106) Google Scholar). For precursor ion scanning resolution of the first quadrupole was set to 1 Da. For operation in the MS/MS mode, the resolution of the first quadrupole was decreased to transmit a window of 4 Da, and the resolution of Q3 was adjusted to 1.5 Da. The daughter ion spectra were used to identify the protein with SEQUEST program (44Yates III, J.R. Eng J.K. McCormack A.L. Schieltz D. Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1109) Google Scholar). Parasite total RNA was isolated using Trizol (Invitrogen) as described (45Kyes S. Pinches R. Newbold C. Mol. Biochem. Parasitol. 2000; 105: 311-315Crossref PubMed Scopus (229) Google Scholar), and RNA was stored in formamide at −80 °C. For Northern blot analysis equal amounts of RNA extracted from synchronized parasite cultures was electrophoresed on 1.2% agarose gels (5 mm guanidine isothiocyanate) (45Kyes S. Pinches R. Newbold C. Mol. Biochem. Parasitol. 2000; 105: 311-315Crossref PubMed Scopus (229) Google Scholar) and vacuum-transferred to a Hybond-XL nylon membrane (AmershamBiosciences). Probes for Northern analysis of pfrpa1 were gel-purified PCR products (see Fig. 6) radiolabeled with [α-32P]dCTP using random primers and Klenow polymerase. Hybridization was performed at 42 °C in UltraHyb (Ambion). Sequence data for pfrpa1 (GenBankTM accession number AL035475) was obtained from the Sanger Center website at www.sanger.ac.uk/Projects/P_falciparum/. Sequencing of P. falciparum chromosome 4 was accomplished as part of the Malaria Genome Project. Preliminary sequence data from the Plasmodium yoellii genome were obtained from the Institute for Genomic Research website (www.tigr.org). This sequencing program is carried on in collaboration with the Naval Medical Research Center. In the course of investigations of P. falciparum promoters by gel retardation assays using dsDNA probes, we detected a dominant nonspecific DNA binding activity in parasite nuclear extracts derived from asynchronously growing cultures. This activity was only observed, however, when probes were end-labeled with T4 polynucleotide kinase which also labels ssDNA molecules. In contrast, when we used Klenow enzyme to fill in 4-base 5′ protrusions at the ends of double-stranded complementary oligonucleotides (ensuring that only double-stranded molecules are labeled), the DNA-protein complex was hardly detected (data not shown). EMSAs using end-labeled single-stranded oligonucleotides showed that this activity was due to the interaction of a nuclear factor with ssDNA. Fig. 1 A shows the interaction between this factor and the radiolabeled 30-base single-stranded oligonucleotide probe 5B1f (the sequence of 5B1f corresponds to a conserved motif found in var gene promoters (46Voss T.S. Thompson J.K. Waterkeyn J. Felger I. Weiss N. Cowman A.F. Beck H.P. Mol. Biochem. Parasitol. 2000; 107: 103-115Crossref PubMed Scopus (62) Google Scholar)). As shown in Fig. 1 B the single-stranded oligonucleotides 5B1f and 5B1rc added at a 20-fold molar excess competed with binding to the labeled probe. However, a double-stranded 155-bp competitor restriction fragment containing the 5B1f sequence (5B1sub5) did not compete for binding. EMSA competition experiments further revealed that the affinity of the nuclear factor was higher for single-stranded polypyrimidine than for polypurine oligomers (Fig. 1 B). In contrast, heterogeneous dsDNA was a much weaker competitor, and yeast tRNA did not compete at all even if added at 2000-fold weight excess. To further investigate for sequence preferences, we used mutated forms of oligonucleotide 5B1f (5B1fmut1–5, see Table I) in gel retardation competition studies. In these oligos consecutive stretches of six nucleotides each were mutated, where A was replaced by G, G replaced by T, and T replaced by C. Whereas oligos 5B1fmut1, 5B1mut4, and 5B1fmut5 competed equally well or even better compared with the wild type sequence 5B1f, the ssDNA-binding factor had a clearly reduced affinity for oligonucleotides 5B1fmut2 and 5B1fmut3 (Fig. 1 C), indicating a certain degree of sequence preference independent of the pyrimidine/purine content. To investigate whether the ssDNA binding activity was present throughout the intra-erythrocytic life cycle, we performed gel retardation experiments using nuclear extracts prepared from synchronously growing cultures. The ssDNA binding activity was absent in mid-ring stage parasites (8–16 h post-invasion (hpi)) (Fig. 2). The activity faintly appeared in young trophozoites (16–24 hpi) and increased to maximal levels in parasites older than 34 hpi. Parasite nuclear extracts derived from the very early ring stage (0–8 hpi) also contained the ssDNA binding activity. A major ssDNA binding activity was also observed in cytoplasmic parasite extracts, but the complexed probe migrated at a slightly different position than the complex formed with the nuclear factor (Fig. 3 A). However, when various protease inhibitors were used during protein isolation and EMSAs, and when using fresh cytoplasmic extracts in gel retardation assays, an additional signal migrating at the same position as the nuclear complex was observed (Fig. 3 B). This suggested identical activities in both subcellular compartments with proteolytic activities in cytosolic extracts acting on the ssDNA-binding factor during protein isolation and gel retardation experiments. In EMSA affinity assays using a variety of different competitor DNAs, the cytoplasmic and nuclear activities behaved identically (Fig. 3 C). These observations supported the assumption that both activities were exerted by the same protein. To investigate this possibility in more detail, we compared limited tryptic digests of UV-cross-linked EMSA reactions by SDS-PAGE (Fig. 4). Without trypsin digestion the major cross-linked complexes in nuclear and cytoplasmic extracts migrated at an equal position (∼65 kDa). The additional larger signal observed in nuclear extracts was probably due to another abundant ssDNA binding activity that was unstable under electrophoresis conditions without UV cross-linking. Furthermore, in both experiments an identical pattern of labeled tryptic fragments was observed indicating that the ssDNA binding activity was retained in tryptic fragments of equal size. Taken together, these findings clearly suggested that the activities present in nuclear and cytoplasmic extracts were identical. Both major ssDNA binding activities from nuclear and cytosolic extracts were purified by affinity purification. Straptavidin-coated magnetic beads with tethered biotinylated 90-base oligonucleotide 5B1Af (a 3-mer of 5B1f) were incubated with crude protein extracts in binding buffer. After washing, bound proteins were eluted with 0.5 and 1 m KCl, and all fractions were analyzed by SDS-PAGE followed by silver staining (data not shown). Testing for ssDNA binding activity using gel retardation revealed that most of the ssDNA binding activity was eluted at 0.5m salt (data not shown). The electrophoretic mobilities of ssDNA-protein complexes formed with crude extracts and purified fractions were identical and are presented in Fig. 5 A. Fig. 5 B shows SDS-PAGE analysis of the 0.5 m cytosolic eluate revealing a dominant band at ∼55 kDa and two additional enriched proteins at 30 and 25 kDa. Similar results were obtained for the nuclear 0.5m eluate. However, the purified cytosolic 55-kDa protein had a slightly smaller size than the nuclear factor (data not shown) which is in agreement with the observed difference in mobility of the corresponding ssDNA-protein complexes in EMSA experiments. Furthermore, SDS-PAGE analysis of UV cross-linked ssDNA-protein complexes obtained with crude extracts and the purified factors revealed a size of ∼65 kDa for each complex (Fig. 5 C). These results strongly suggested that the 55-kDa factor was responsible for ssDNA binding because the size difference of 10 kDa observed between the purified protein alone and the UV cross-linked ssDNA-protein complexes was accounted for by the covalent attachment of the 30-base oligonucleotide 5" @default.
• W1997091164 created "2016-06-24" @default.
• W1997091164 creator A5013432533 @default.
• W1997091164 creator A5019506578 @default.
• W1997091164 creator A5032034269 @default.
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• W1997091164 date "2002-05-01" @default.
• W1997091164 modified "2023-09-27" @default.
• W1997091164 title "Plasmodium falciparum Possesses a Cell Cycle-regulated Short Type Replication Protein A Large Subunit Encoded by an Unusual Transcript" @default.
• W1997091164 cites W1517046087 @default.
• W1997091164 cites W1550656265 @default.
• W1997091164 cites W1554850792 @default.
• W1997091164 cites W1589132235 @default.
• W1997091164 cites W1590134197 @default.
• W1997091164 cites W1590500446 @default.
• W1997091164 cites W1625430625 @default.
• W1997091164 cites W1659681861 @default.
• W1997091164 cites W1778599501 @default.
• W1997091164 cites W1899289062 @default.
• W1997091164 cites W1965179505 @default.
• W1997091164 cites W1977835715 @default.
• W1997091164 cites W1983134926 @default.
• W1997091164 cites W1985794196 @default.
• W1997091164 cites W1990387262 @default.
• W1997091164 cites W1991547782 @default.
• W1997091164 cites W1991686350 @default.
• W1997091164 cites W1995806503 @default.
• W1997091164 cites W1997007140 @default.
• W1997091164 cites W1999632568 @default.
• W1997091164 cites W2002496482 @default.
• W1997091164 cites W2002565384 @default.
• W1997091164 cites W2002665761 @default.
• W1997091164 cites W2004615780 @default.
• W1997091164 cites W2005064689 @default.
• W1997091164 cites W2006795488 @default.
• W1997091164 cites W2007910316 @default.
• W1997091164 cites W2013817482 @default.
• W1997091164 cites W2017175281 @default.
• W1997091164 cites W2020251171 @default.
• W1997091164 cites W2026933272 @default.
• W1997091164 cites W2030826575 @default.
• W1997091164 cites W2035746057 @default.
• W1997091164 cites W2040159897 @default.
• W1997091164 cites W2040378961 @default.
• W1997091164 cites W2045264503 @default.
• W1997091164 cites W2047734632 @default.
• W1997091164 cites W2048255483 @default.
• W1997091164 cites W2055043387 @default.
• W1997091164 cites W2055462773 @default.
• W1997091164 cites W2057260776 @default.
• W1997091164 cites W2059572899 @default.
• W1997091164 cites W2060850037 @default.
• W1997091164 cites W2062028908 @default.
• W1997091164 cites W2064045867 @default.
• W1997091164 cites W2064986560 @default.
• W1997091164 cites W2067154171 @default.
• W1997091164 cites W2070776091 @default.
• W1997091164 cites W2072475400 @default.
• W1997091164 cites W2078882775 @default.
• W1997091164 cites W2079471238 @default.
• W1997091164 cites W2080977505 @default.
• W1997091164 cites W2085326030 @default.
• W1997091164 cites W2087917314 @default.
• W1997091164 cites W2088090805 @default.
• W1997091164 cites W2090585008 @default.
• W1997091164 cites W2098822637 @default.
• W1997091164 cites W2100584834 @default.
• W1997091164 cites W2108091759 @default.
• W1997091164 cites W2123527421 @default.
• W1997091164 cites W2139768549 @default.
• W1997091164 cites W2142844417 @default.
• W1997091164 cites W2149389239 @default.
• W1997091164 cites W2168166289 @default.
• W1997091164 cites W2171775098 @default.
• W1997091164 cites W838635636 @default.
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