Fission Yeast Mcm10p Contains Primase Activity
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m512997200
ISSN1083-351X
Autores Tópico(s)RNA modifications and cancer
ResumoAlthough Mcm10p is a conserved essential component in eukaryotes required for both the initiation and elongation of DNA chains, its biochemical properties are unknown. Here, we report that the Schizosaccharomyces pombe fission yeast Mcm10 protein contains primase activity. Primases are enzymes that synthesize RNA primers on single-stranded DNA templates that are extended by DNA polymerases. In keeping with this property, Mcm10p supported oligoribonucleotide synthesis of short RNA primers (preferentially initiating synthesis on a dT template) that were extended with dATP by Escherichia coli DNA polymerase I. The C terminus of Mcm10p synthesized RNA, but less efficiently than the full-length protein at low rNTP levels. Mcm10p homologs contain a C-terminal motif found in proteins that polymerize nucleotides. A point mutant within this motif of S. pombe Mcm10p was defective in primer synthesis in vitro, and this mutant failed to support growth in vivo, suggesting that the primase activity of Mcm10p may be essential for cell viability. Although Mcm10p is a conserved essential component in eukaryotes required for both the initiation and elongation of DNA chains, its biochemical properties are unknown. Here, we report that the Schizosaccharomyces pombe fission yeast Mcm10 protein contains primase activity. Primases are enzymes that synthesize RNA primers on single-stranded DNA templates that are extended by DNA polymerases. In keeping with this property, Mcm10p supported oligoribonucleotide synthesis of short RNA primers (preferentially initiating synthesis on a dT template) that were extended with dATP by Escherichia coli DNA polymerase I. The C terminus of Mcm10p synthesized RNA, but less efficiently than the full-length protein at low rNTP levels. Mcm10p homologs contain a C-terminal motif found in proteins that polymerize nucleotides. A point mutant within this motif of S. pombe Mcm10p was defective in primer synthesis in vitro, and this mutant failed to support growth in vivo, suggesting that the primase activity of Mcm10p may be essential for cell viability. DNA replication occurs through a complex series of reactions that are mechanistically coordinated at the replication fork. Studies of model DNA replication systems indicate that origin recognition by an initiator protein permits assembly and activation of the replicative helicase at the origin. Helicases unwind duplex DNA to generate a single-strand template on which primases can initiate synthesis of RNA primers that are 4-20 nucleotides in length and that are extended by DNA polymerases to make Okazaki fragments (1Frick D.N. Richardson C.C. Annu. Rev. Biochem. 2001; 70: 39-80Crossref PubMed Scopus (305) Google Scholar). Primases are physically coupled to the replicative DNA helicases and DNA polymerases, which translocate together through the duplex, resulting in DNA unwinding coincident with synthesis of RNA primers and their extension with deoxynucleotides. In eukaryotes, the Mcm proteins are essential replication factors that were identified as proteins required for minichromosomal maintenance in a genetic screen for mutants defective in initiation of replication (2Tye B.K. Annu. Rev. Biochem. 1999; 68: 649-686Crossref PubMed Scopus (516) Google Scholar). Six of these members are sequence-related proteins, Mcm2-7, which interact to form a hexameric complex. Although a substantial body of data suggests that the Mcm2-7p complex acts as the replicative helicase (3Blow J.J. Dutta A. Nat. Rev. Mol. Cell Biol. 2005; 6: 476-486Crossref PubMed Scopus (533) Google Scholar), helicase activity has been detected only in the Mcm4-6-7 subcomplex (4Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 5Kaplan D.L. Davey M.J. O'Donnell M. J. Biol. Chem. 2003; 278: 49171-49182Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 6Lee J.-L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 54-59Crossref PubMed Scopus (159) Google Scholar). Prior to the initiation of replication, the Mcm2-7 complex associates with the initiator at replication origins. At the G1/S transition, both S phase cyclin-dependent kinase and Cdc7p-Dbf4p kinase activities rise, promoting the maturation of the pre-replicative complex to the preinitiation complex (7Zou L. Stillman B. Science. 1998; 280: 593-596Crossref PubMed Scopus (275) Google Scholar). Phosphorylation of the chromatin-bound Mcm2-7 complex by the Cdc7-Dbf4 kinase (8Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (249) Google Scholar) is thought to trigger the binding of Mcm10p, Cdc45p, and the GINS complex with origins prior to the initiation of replication (9Kanemaki M. Sanchez-Diaz A. Gambus A. Labib K. Nature. 2003; 423: 720-724Crossref PubMed Scopus (215) Google Scholar, 10Takayama Y. Kamimura Y. Okawa M. Muramatsu S. Sugino A. Araki H. Genes Dev. 2003; 17: 1153-1165Crossref PubMed Scopus (283) Google Scholar). Although Mcm10p was identified in the same genetic screen as the Mcm2-7 proteins, their amino acid sequences are unrelated. Studies in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, and Xenopus laevis indicate that Mcm10p is an essential conserved replication component. Mutations in mcm10 result in a marked reduction in the initiation of replication as well as a delay in S phase after hydroxyurea arrest, suggesting that Mcm10p is involved in initiation as well as elongation steps of replication (11Nasmyth K. Nurse P. Mol. Gen. Genet. 1981; 182: 119-124Crossref PubMed Scopus (226) Google Scholar, 12Solomon N.A. Wright M.B. Chang S. Buckley A.M. Dumas L.B. Gaber R.F. Yeast. 1992; 8: 273-289Crossref PubMed Scopus (58) Google Scholar, 13Merchant A.M. Kawasaki Y. Chen Y. Lei M. Tye B. Mol. Cell. Biol. 1997; 17: 3261-3271Crossref PubMed Scopus (119) Google Scholar, 14Kawasaki Y. Hiraga S.-I. Sugino A. Genes Cells. 2000; 5: 975-989Crossref PubMed Scopus (67) Google Scholar). Mcm10p was shown recently to associate with origins in a cell cycle- and Mcm4p-dependent manner and with origin distal sequences during S phase (15Ricke R. Bielinsky A.-K. Mol. Cell. 2004; 16: 173-185Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Mcm10p interacts genetically and biochemically with other replication factors, including the Mcm2-7 complex; origin recognition complex; Cdc45p; Dna2p; the subunits of DNA polymerases (pol) 3The abbreviations used are: pol, DNA polymerase; SpMcm10p, S. pombe Mcm10p; ssDNA, single-stranded DNA; NTD, nucleotide transfer domain; MES, 4-morpholineethanesulfonic acid. α, δ, and ϵ; and S. pombe Dfp1p-Hsk1p (Cdc7p-Dbf4p) (14Kawasaki Y. Hiraga S.-I. Sugino A. Genes Cells. 2000; 5: 975-989Crossref PubMed Scopus (67) Google Scholar, 16Homesley L. Lei M. Kawasaki Y. Sawyer S. Christensen T. Tye B.K. Genes Dev. 2000; 14: 913-926PubMed Google Scholar, 17Liu Q. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 1615-1624Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18Liang D.T. Forsburg S.L. Genetics. 2001; 159: 471-486Crossref PubMed Google Scholar, 19Hart E.A. Bryant J.A. Moore K. Aves S.J. Curr. Genet. 2002; 41: 342-348Crossref PubMed Scopus (29) Google Scholar, 20Araki Y. Kawasaki Y. Sasanuma H. Tye B.K. Sugino A. Genes Cells. 2003; 8: 465-480Crossref PubMed Scopus (37) Google Scholar, 21Lee J.-K. Seo Y.-S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2334-2339Crossref PubMed Scopus (74) Google Scholar). In both yeast and Xenopus, Mcm10p is required for the loading of Cdc45p onto chromatin (22Wohlschlegel J.A. Dhar S.K. Prokhorova T.A. Dutta A. Walter J.C. Mol. Cell. 2002; 9: 233-240Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 23Gregan J. Lindner K. Brimage L. Franklin R. Namdar M. Hart E.A. Aves S.J. Kearsey S.E. Mol. Biol. Cell. 2003; 14: 3876-3887Crossref PubMed Scopus (90) Google Scholar, 24Sawyer S.L. Cheng I.H. Chai W. Tye B.K. J. Mol. Biol. 2004; 340: 195-202Crossref PubMed Scopus (66) Google Scholar). Previously, we reported that, in vitro, S. pombe Mcm10p (amino acids 1-593; SpMcm10p) binds preferentially to single-stranded DNA (ssDNA) and to the large subunit of the pol α-primase complex and activates its DNA synthetic activity (25Fien K. Cho Y.-S. Lee J.-K. Raychaudhuri S. Tappin I. Hurwitz J. J. Biol. Chem. 2004; 279: 16144-16153Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In addition, we found that Mcm10p facilitates the binding of the pol α-primase complex to primed DNA and forms a stable ternary complex, suggesting that Mcm10p recruits the pol α-primase complex to template DNA. In keeping with these in vitro findings, Mcm10p was shown recently to stabilize the large subunit of the pol α-primase complex in S. cerevisiae (15Ricke R. Bielinsky A.-K. Mol. Cell. 2004; 16: 173-185Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) as well as the chromatin association of the pol α-primase complex in S. pombe (26Yang X. Gregan J. Lindner K. Young H. Kearsey S.E. BMC Mol. Biol. 2005; 6: 13Crossref PubMed Scopus (38) Google Scholar). In this study, we demonstrate that full-length SpMcm10p (amino acids 1-593) and its C-terminal fragment (amino acids 416-593) contain primase activity that catalyzes the synthesis of oligoribonucleotides that are extended by Escherichia coli pol I. Primase activity both co-sedimented and co-eluted with these full-length and truncated proteins, although their hydrodynamic properties differ significantly. The C-terminal domain of Mcm10p (and its homologs in other organisms) contains conserved acidic residues characteristic of proteins that polymerize nucleotides (the nucleotide transfer domain (NTD)) (27Aravind L. Koonin E.V. Nucleic Acids Res. 1999; 27: 1609-1618Crossref PubMed Scopus (276) Google Scholar). A single amino acid substitution in one of these residues in full-length Mcm10p greatly reduced its primase activity (>10-fold). Further analysis of single amino acid substitutions of these conserved acidic residues suggests that they are essential for viability in yeast. Preparation of Mcm10 Proteins—Mcm10 proteins were overproduced in E. coli strain BL21(DE3)RIL containing various pET28a-MCM10 expression plasmids. Construction of plasmids, growth and lysis of cells, and purification of proteins by nickel-nitrilotriacetic acid affinity were as described (25Fien K. Cho Y.-S. Lee J.-K. Raychaudhuri S. Tappin I. Hurwitz J. J. Biol. Chem. 2004; 279: 16144-16153Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) with the following exceptions. To obtain higher yields of protein, cells were grown in medium (4 liter) containing 100 ml of 10× M9 medium (10 g/liter NH4Cl, 30 g/liter KH2PO4, and 60 g/liter Na2HPO4), 200 ml of 5× medium (50 g/liter Tryptone, 25 g/liter yeast extract, and 25 g/liter NaCl), 20 ml of 20% glucose, 1 ml of 1 m MgSO4, and 40 μg/ml each kanamycin and chloramphenicol. Cell lysates were centrifuged at 10,000 rpm for 10 min; the supernatant was adjusted to 50% saturation with (NH4)2SO4; and the suspension was stirred for 20 min at 4 °C. The precipitate was collected at 18,000 rpm for 60 min and dissolved in lysate buffer. Mcm10p was bound to nickel-nitrilotriacetic acid beads, and the imidazole eluate was adjusted to 250 mm NaCl with Buffer A (25 mm MES-KOH (pH 6.5), 5% glycerol, 10 mm magnesium acetate, 0.1% Nonidet P-40, 2 mm EDTA, 1 mm dithiothreitol, 20 μm ZnSO4, and 1 mm phenylmethylsulfonyl fluoride). This material was loaded onto a 1-ml Mono S HR 5/50 column equilibrated with Buffer A containing 200 mm NaCl and eluted with a 6-ml gradient of 200-800 mm NaCl. Mcm10p eluted between ∼400 and 500 mm NaCl (yield of 2.0 mg of protein). A portion of the purified Mono S fraction (0.40 mg) (see Fig. 1A, lane 1) was used for subsequent analysis. Mcm10p-(1-303) and Mcm10p-(416-593) were purified similarly, except that Mcm10p-(416-593) was isolated using a 1-ml Mono Q HR 5/50 column in place of the Mono S column. The yields of protein were 0.5 and 1 mg from 2 liter of medium, respectively. Primase Assay of Oligoribonucleotide Synthesis—Primase activity was determined by measuring the amount of oligoribonucleotide synthesized in the presence of a DNA template. Standard reaction mixtures (10 μl) contained 1.0 μm (dT)50, 40 mm Tris-HCl (pH 7.4), 10 mm magnesium acetate, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin, 0.1 mm ATP, 25 μCi of [α-32P]ATP, and various levels of full-length Mcm10p or truncated derivatives. Reaction mixtures containing either 1.0 μm (dC)50 or 0.2 pmol of M13mp18 ssDNA were similar to those described for (dT)50, except that they contained either 0.1 mm GTP and 25 μCi of [α-32P]GTP or 0.1 mm each ATP, GTP, CTP, and UTP and 25 μCi of [α-32P]ATP, respectively. Reactions were incubated at 37 °C for 40 min unless specified otherwise. The reactions in Fig. 4 were stopped at the times indicated by heat inactivation at 95 °C for 5 min and chilled on ice. Calf intestine alkaline phosphatase (1 unit) was added, and the mixture was incubated for 30 min at 37 °C. Reactions were terminated by the addition of 3 μl of sequencing dye (98% formamide, 10 mm EDTA (pH 8.0), 0.1% xylene cyanol, and 0.1% bromphenol blue), heated to 98 °C for 5 min, and separated on a 25% denaturing polyacrylamide sequencing gel containing 7 m urea. Labeled oligoribonucleotide products were visualized using a Fuji BAS 1000 bioimaging analyzer and quantitated from a phosphorimage of the gel. Primer-dependent DNA Synthesis Assay—Oligoribonucleotides synthesized by full-length Mcm10p, Mcm10p-(416-593), or Mcm10p-(1-303) were assayed by measuring their ability to serve as primers for the E. coli pol I large fragment (Klenow). The conditions used were the same as those described for the primase-catalyzed oligoribonucleotide synthesis assay, except for the following additions: 0.1 mm dATP, 0.08 unit of Klenow fragment, 1.5 μCi of [α-32P]dATP in place of radiolabeled ATP, and 0.1 μm (dT)300 in place of (dT)50. The longer dT template supported ∼10 times more dAMP incorporation than the shorter template. Reaction mixtures were incubated for 40 min at 37 °C, after which half of the reaction mixture was mixed with 10 μl of alkaline loading dye (0.5 m NaOH, 7mm EDTA (pH 8.0), 0.5% Ficoll, 0.1% xylene cyanol, and 0.1% bromphenol blue), and DNA products were separated on 2% alkaline agarose gels. The amount of nucleotide incorporated into DNA was measured as described (25Fien K. Cho Y.-S. Lee J.-K. Raychaudhuri S. Tappin I. Hurwitz J. J. Biol. Chem. 2004; 279: 16144-16153Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) or by using a phosphorimage of the gel. Computer-assisted Analysis of Amino Acid Sequences—The data used in this study were from the NCBI Database; the protein sets were encoded in publicly available completely sequenced genomes. Initially, we used P-BLAST to identify Mcm10p amino acid sequences from different organisms with blast hits above a certain expectation value (e-value). Eleven full-length Mcm10p amino acid sequences from different divergent organisms found by the BLAST search were then aligned using the ClustalW program. This program produces biologically meaningful multiple sequence alignments of divergent sequences. The ClustalW program allows putative motifs to be identified because of the calculated best match for a large group of related selected sequences and lines them up so that the identities, similarities, and differences can be seen. The following is a list of the Mcm10 protein sequences used in the alignment and their accession numbers: X. laevis (frog) Mcm10p (AAG33858), Homo sapiens MCM10 (BAB18723), S. cerevisiae (budding yeast) Mcm10p (CAA86128), S. pombe (fission yeast) Mcm10p (BAA24935), Rattus norvegicus (rat) MCM10 (XP_225570), D. melanogaster (fly) MCM10 (AAF53976), Neurospora crassa (bread mold) Mcm10-like protein (EAA31137), Mus musculus (mouse) MCM10 (NP_081566), Caenorhabditis elegans (worm) MCM10-like protein (CAB57900), Anopheles gambiae (malaria mosquito) Mcm10-like protein (EAA10289), and Plasmodium yoelii yoelii (rodent malaria) Mcm10-like protein (EAA17050). Construction of Mcm10p Point Mutations—The MCM10 shuffle strain used in this study was ILY251 (MATa leu2-3,112, ura3-52, mcm10::hisG (pRS315-MCM10-URA3)) (a gift from Bik Tye), and the wild-type S. cerevisiae Mcm10p strain used was JPY9 (MATa his3-D200, ura3-52, leu2-D1, trp1D63, gal4D11). Plasmids pET-Mcm10p(E586A), pET-Mcm10p(D587A), and pET-Mcm10p(D588A), expressing Mcm10p containing Ala in place of Glu at position 586 or in place of Asp at position 587 or 588, respectively, were constructed by PCR mutagenesis of the pET28a-MCM10 plasmid. The presence of the mutated site was screened by restriction digestion with BstUI and by sequencing to verify specific changes. Mutated plasmids were digested either with NdeI and NotI or with BamHI and XhoI, and 1.7-kb fragments were cloned either into the yeast-E. coli shuttle vector pRSSGS415 (LEU2) containing the SGS1 promoter (a gift from Steve Brill) and digested with NdeI and NotI or into the vector pRSADH425 (LEU2) containing the strong alcohol dehydrogenase promoter and digested with BamHI and XhoI. Both shuttle vectors expressing mutant and SpMcm10p were examined in the plasmid shuffle assay. Mcm10p Catalyzes Template-dependent Synthesis of Oligoribonucleotides—We examined whether SpMcm10p and its N- and C-terminally truncated derivatives, Mcm10p-(1-303) and Mcm10p (416-597), catalyze template-dependent oligoribonucleotide synthesis. The purity of these preparations, which were expressed in E. coli and purified, is shown in Fig. 1A (lanes 1-3). These protein preparations were screened for their ability to synthesize small oligoribonucleotides using [α-32P]ATP and single-stranded (dT)50 as the template. In the presence of ATP (100 μm), (dT)50, and full-length SpMcm10p, RNA products 4-18 nucleotides in length were produced (Fig. 1B, lanes 5 and 6). In the absence of an ssDNA template or in the presence of a low level of ATP (0.01 μm), oligoribonucleotide synthesis was not observed (Fig. 1B, lanes 2 and 3 and lanes 8 and 9, respectively). Quantitation of the amount of RNA formed indicated that the purified SpMcm10p supported incorporation of 0.3 pmol of AMP/min/pmol of protein at 37 °C. This level of activity is comparable with that observed with the phage T7 primase T7g4p (T7 gene 4p; 0.3 pmol of CMP incorporated per min/pmol of protein) (28Lee S.J. Richardson C.C. J. Biol. Chem. 2001; 276: 49419-49426Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). To identify the region in SpMcm10p responsible for primase activity, we examined two truncated derivatives of SpMcm10p in the oligoribonucleotide synthesis assay. As shown in Fig. 1C, Mcm10p-(416-593) supported oligoribonucleotide synthesis (lanes 9-14), whereas Mcm10p-(1-303) did not (lanes 19 and 20). Like full-length SpMcm10p, Mcm10p-(416-593) required added ssDNA for RNA synthesis (lanes 15-17). Whereas full-length Mcm10p supported oligoribonucleotide synthesis in the presence of ATP at 2.5 μm and higher, the truncated protein Mcm10p-(416-593) required at least 25 μm ATP. Significant differences in the length of RNA products formed by full-length Mcm10p and Mcm10p-(416-593) were noted. At all levels of protein tested, full-length Mcm10p synthesized products 5-18 nucleotides in length, whereas high levels of Mcm10p-(416-593) accumulated significantly longer products (Fig. 1C). Quantitative analysis of the RNA products formed at different levels of added ATP indicated that full-length Mcm10p had a higher specific activity compared with Mcm10p-(416-593) in the presence of 2 pmol of protein (supplemental Fig. 1A), whereas in the presence of 5 pmol of protein, the truncated derivative was five times more active than full-length Mcm10p (Fig. 1, compare B, lane 6, and C, lane 13). These results indicate that the residues between positions 416 and 593 of Mcm10p encode an RNA polymerase activity, but that the N-terminal regions of Mcm10p may play a role in regulating both the length and efficiency of oligoribonucleotide synthesis at low ATP levels. A recent study with the E. coli DnaG primase catalytic domain showed similarly that this region synthesizes primers whose abundance and lengths differ from those synthesized by the full-length primase (29Corn J.E. Pease P. Hura G. Berger J. Mol. Cell. 2005; 20: 391-401Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We titrated Mcm10p-(416-593) in the direct primase assays and found that the activity curve was sigmoidal with respect to protein concentration, in contrast to that observed with full-length Mcm10p. The possible significance of this result is discussed further below. RNA Primers Formed by Mcm10p Support DNA Synthesis—The biological role of oligoribonucleotides synthesized by primase is to serve as primers for DNA polymerases that are incapable of initiating DNA chains de novo. To evaluate whether oligoribonucleotides formed by Mcm10p support DNA synthesis, we used the E. coli pol I-primase-coupled assay (30Kaguni L.S. Lehman I.R. Biochim. Biophys. Acta. 1988; 950: 87-101Crossref PubMed Scopus (44) Google Scholar). In this assay, which was carried out with (dT)300, the Klenow fragment-catalyzed incorporation of [α-32P]dATP is totally dependent on the oligoriboadenylate primers formed by primase. As shown in Fig. 2 (A and B), both full-length Mcm10p and the truncated derivative supported dAMP incorporation and formation of DNA chains 1-1.5 kb in length. Poly(dA) synthesis required the addition of ATP, (dT)300, either full-length Mcm10p or Mcm10p-(416-593), and the Klenow fragment (data showing the latter requirement not shown). In keeping with the results shown in Fig. 1C, full-length Mcm10p (amino acids 1-593) supported significantly more DNA synthesis at ∼10-fold lower levels of ATP compared with Mcm10p-(416-593) (Fig. 2B). The primase activity of the Mcm10p derivatives and the S. pombe p48-p58 primase complex (normally associated with the p180-p70 complex of pol α-primase) were compared using the coupled Klenow extension assay. As shown in Fig. 2C, full-length Mcm10p and Mcm10p-(416-593) were 5- and 12-fold less active, respectively, than the p48-p58 complex. These findings indicate that the Klenow fragment extends oligoriboadenylate chains formed by the Mcm10 proteins. When the extension assay was carried out in reactions containing the S. pombe pol α-primase p180-p70 subcomplex and the S. pombe pol δ or δ holoenzyme (including S. pombe replication factor C and S. pombe proliferating cell nuclear antigen) in lieu of the E. coli Klenow fragment, low but significant DNA synthesis was detected (supplemental Table 1). Physical Properties of Full-length Mcm10p, Mcm10p-(1-303), and Mcm10p-(416-593)—To characterize the hydrodynamic properties of full-length Mcm10p and its truncated derivatives and to establish whether they contain intrinsic primase activity, purified full-length Mcm10p, Mcm10p-(1-303), and Mcm10p-(416-593) were subjected to Superdex 200 gel filtration chromatography and glycerol gradient centrifugation. Fractions eluted from the size column were examined for primase activity in the coupled polymerase assay. Both full-length Mcm10p and Mcm10p-(416-593) eluted as a single protein peak that was largely coincident with the peak of primase activity, whereas fractions containing Mcm10p-(1-303) were devoid of this activity (Fig. 3A). The results obtained with glycerol gradient fractions indicated similarly that full-length Mcm10p and Mcm10p-(416-593) both eluted with a single peak of activity that was coincident with the peak of primase activity, whereas fractions containing Mcm10p-(1-303) were devoid of activity (Fig. 3B) (data not shown). These findings suggest that both full-length Mcm10p and Mcm10p-(416-593) intrinsically contain primase activity. Full-length Mcm10p displayed distinct properties in the two different sizing procedures used. The activity eluted from the gel filtration column with a relative molecular mass of 220 kDa, whereas the activity sedimented through a glycerol gradient at 70 kDa. The reason for the differential behavior during these two sizing steps is likely due to an irregularity in shape, which can cause a protein to be more excluded than spherical standards during gel filtration and sediment more slowly during centrifugation (31Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar). The Siegel-Monty equation (31Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar), which employs both the sedimentation value and Stokes radius, was used to calculate the apparent molecular mass of Mcm10p as well as its truncated derivatives (Fig. 3C). The apparent molecular masses of Mcm10p (120 kDa) and Mcm10p-(1-303) (74 kDa) are close to the values expected for a dimer (133 and 70 kDa, respectively), whereas that of Mcm10p-(416-593) (26 kDa) was less than halfway between that of a monomer (19 kDa) and a dimer (38 kDa). These data suggest that Mcm10p is a dimeric structure and that its dimerization may be influenced by the N terminus. We titrated Mcm10p-(416-593) in the direct and Klenow extension primase assays and found that the activity was not linear as found with Mcm10p. Instead, the activity observed was sigmoidal with respect to protein concentration. Although the reasons for this property are unclear, it should be noted that wild-type primase appears to be a dimer and that the region removed in this truncation (the N terminus) appears to be required for the dimerization. An interesting possibility is that, in the absence of the N terminus, high concentrations of truncated protein can substitute for dimer formation. Mcm10p-catalyzed Oligoribonucleotide Synthesis Is Influenced by the Template—Primases usually synthesize oligoribonucleotides complementary to template ssDNAs (1Frick D.N. Richardson C.C. Annu. Rev. Biochem. 2001; 70: 39-80Crossref PubMed Scopus (305) Google Scholar). However, prokaryotic primases support higher rates of oligoribonucleotide synthesis in the presence of specific DNA sequences called primase recognition sites. Although eukaryotic primases lack stringent requirements for such sites, they display sequence preferences. Primase initiation site selection depends on the reaction conditions used and appears to be more random at high rNTP concentrations and/or in the presence of Mn2+. For this reason, oligoribonucleotide synthesis with Mcm10p and the S. pombe p48-p58 primase complex was examined in the presence of (dT)50, (dC)50, and M13mp18 ssDNA at various rNTP levels (only with Mg2+). We examined the rate of oligoriboadenylate synthesis in the presence of (dT)50 and the S. pombe p48-p58 primase complex or full-length Mcm10p (Fig. 4A). In these experiments, the concentration of ATP was 100 μm, and the products formed were subjected to calf intestine alkaline phosphatase digestion. This treatment altered both the size and distribution of the oligoriboadenylate products formed (compare Figs. 1B and 4A) and prevented the detection of products smaller than 8 nucleotides. The aberrant migration of small RNAs due to calf intestine alkaline phosphatase treatment has been noted previously (32Cha T.A. Alberts B.M. Biochemistry. 1990; 29: 1791-1798Crossref PubMed Scopus (37) Google Scholar) (Fig. 4F). These conditions facilitated the synthesis by the p48-p58 primase complex of short RNA products, which were not detected in reactions containing 25 μm ATP (compare Fig. 4A and supplemental Fig. 1B). The rate of oligoribonucleotide formation by Mcm10p was slower than that by p48-p58 (Fig. 4, A and C). In the presence of p48-p58, the synthesis of oligoriboadenylate was detected 1 min after enzyme addition and plateaued after 10 min of incubation. In contrast, product formation with Mcm10p showed a pronounced lag of 5 min and then increased linearly up to 40 min. The size distribution of the products formed differed as well; products formed with Mcm10p were more varied in length than those synthesized by the p48-p58 complex. Even at a high GTP concentration (100 μm), reactions that contained (dC)50 produced low levels of oligomers in the presence of Mcm10p (Fig. 4, B and D), in keeping with observations made at lower nucleotide levels (supplemental Fig. 1B). However, at the higher GTP concentration, p48-p58 produced substantial levels of oligomer considerably longer in length than that formed at lower GTP levels (compare Fig. 4D and supplemental Fig. 1B). These data indicate that Mcm10p initiates primer synthesis preferentially with ATP rather than GTP. In general, primases do not initiate RNA chains with pyrimidine nucleotides, and in keeping with this property, dTTP incorporation was not observed in the coupled DNA synthesis reactions using poly(dA) as the template (in the presence of UTP and [α-32P]dTTP) with either Mcm10p or p48-p58 (data not shown). We found that the primers synthesized by Mcm10p were not efficiently extended farther in the presence of dATP and the S. pombe pol α-primase p180-p70 subcomplex (supplemental Fig. 1B and supplemental Table 1), whereas the oligo(G) product formed by the p48-p58 complex was extended by dGTP addition in the presence of the S. pombe pol α-primase p180-p70 subcomplex. These results suggest that primases may hand off primers more efficiently to one DNA polymerase compared with another. We also examined RNA products formed in reactions containing M13mp18 ssDNA as the template in the presence of the four rNTPs ([α-32P]ATP) at various rNTP levels. In these experiments, all products formed were subjected to calf intestine alkaline phosphatase digestion (Fig. 4, E and F). The major products synthes
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