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Mol Cell Biol. 1994 Feb; 14(2): 923–933.
doi: 10.1128/mcb.14.2.923
PMCID: PMC358447
PMID: 8289832

The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

M Foiani, F Marini, D Gamba, G Lucchini, and P Plevani
Author information Copyright and License information PMC Disclaimer

Abstract

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

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Selected References

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  • Araki H, Hamatake RK, Johnston LH, Sugino A. DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4601–4605. [PMC free article] [PubMed] [Google Scholar]
  • Araki H, Ropp PA, Johnson AL, Johnston LH, Morrison A, Sugino A. DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae. EMBO J. 1992 Feb;11(2):733–740. [PMC free article] [PubMed] [Google Scholar]
  • Bell SP, Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. [PubMed] [Google Scholar]
  • Brill SJ, Stillman B. Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 1991 Sep;5(9):1589–1600. [PubMed] [Google Scholar]
  • Brooke RG, Dumas LB. Reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex in vitro. The 86-kDa subunit facilitates but is not required for complex formation. J Biol Chem. 1991 Jun 5;266(16):10093–10098. [PubMed] [Google Scholar]
  • Brooke RG, Singhal R, Hinkle DC, Dumas LB. Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities. J Biol Chem. 1991 Feb 15;266(5):3005–3015. [PubMed] [Google Scholar]
  • Budd M, Campbell JL. Temperature-sensitive mutations in the yeast DNA polymerase I gene. Proc Natl Acad Sci U S A. 1987 May;84(9):2838–2842. [PMC free article] [PubMed] [Google Scholar]
  • Budd ME, Wittrup KD, Bailey JE, Campbell JL. DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):365–376. [PMC free article] [PubMed] [Google Scholar]
  • Budd ME, Campbell JL. DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jan;13(1):496–505. [PMC free article] [PubMed] [Google Scholar]
  • Bueno A, Russell P. Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. EMBO J. 1992 Jun;11(6):2167–2176. [PMC free article] [PubMed] [Google Scholar]
  • Collins KL, Kelly TJ. Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase. Mol Cell Biol. 1991 Apr;11(4):2108–2115. [PMC free article] [PubMed] [Google Scholar]
  • Collins KL, Russo AA, Tseng BY, Kelly TJ. The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication. EMBO J. 1993 Dec;12(12):4555–4566. [PMC free article] [PubMed] [Google Scholar]
  • Cotterill S, Lehman IR, McLachlan P. Cloning of the gene for the 73 kD subunit of the DNA polymerase alpha primase of Drosophila melanogaster. Nucleic Acids Res. 1992 Aug 25;20(16):4325–4330. [PMC free article] [PubMed] [Google Scholar]
  • Cross FR. DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4675–4684. [PMC free article] [PubMed] [Google Scholar]
  • Dasso M, Newport JW. Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus. Cell. 1990 Jun 1;61(5):811–823. [PubMed] [Google Scholar]
  • Diffley JF. Early events in eukaryotic DNA replication. Trends Cell Biol. 1992 Oct;2(10):298–303. [PubMed] [Google Scholar]
  • Diffley JF, Cocker JH. Protein-DNA interactions at a yeast replication origin. Nature. 1992 May 14;357(6374):169–172. [PubMed] [Google Scholar]
  • Din S, Brill SJ, Fairman MP, Stillman B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 1990 Jun;4(6):968–977. [PubMed] [Google Scholar]
  • Dingwall C, Laskey RA. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. [PubMed] [Google Scholar]
  • Enoch T, Nurse P. Coupling M phase and S phase: controls maintaining the dependence of mitosis on chromosome replication. Cell. 1991 Jun 14;65(6):921–923. [PubMed] [Google Scholar]
  • Epstein CB, Cross FR. CLB5: a novel B cyclin from budding yeast with a role in S phase. Genes Dev. 1992 Sep;6(9):1695–1706. [PubMed] [Google Scholar]
  • Fangman WL, Brewer BJ. A question of time: replication origins of eukaryotic chromosomes. Cell. 1992 Oct 30;71(3):363–366. [PubMed] [Google Scholar]
  • Foiani M, Cigan AM, Paddon CJ, Harashima S, Hinnebusch AG. GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3203–3216. [PMC free article] [PubMed] [Google Scholar]
  • Foiani M, Santocanale C, Plevani P, Lucchini G. A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jul;9(7):3081–3087. [PMC free article] [PubMed] [Google Scholar]
  • Forsburg SL, Nurse P. Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Annu Rev Cell Biol. 1991;7:227–256. [PubMed] [Google Scholar]
  • Fotedar R, Roberts JM. Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. EMBO J. 1992 Jun;11(6):2177–2187. [PMC free article] [PubMed] [Google Scholar]
  • Francesconi S, Longhese MP, Piseri A, Santocanale C, Lucchini G, Plevani P. Mutations in conserved yeast DNA primase domains impair DNA replication in vivo. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3877–3881. [PMC free article] [PubMed] [Google Scholar]
  • Hartwell LH. Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J Mol Biol. 1971 Jul 14;59(1):183–194. [PubMed] [Google Scholar]
  • Hartwell LH. Sequential function of gene products relative to DNA synthesis in the yeast cell cycle. J Mol Biol. 1976 Jul 15;104(4):803–817. [PubMed] [Google Scholar]
  • Hartwell LH, Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. [PMC free article] [PubMed] [Google Scholar]
  • Hartwell LH, Weinert TA. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. [PubMed] [Google Scholar]
  • Hennessy KM, Clark CD, Botstein D. Subcellular localization of yeast CDC46 varies with the cell cycle. Genes Dev. 1990 Dec;4(12B):2252–2263. [PubMed] [Google Scholar]
  • Hereford LM, Hartwell LH. Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. J Mol Biol. 1974 Apr 15;84(3):445–461. [PubMed] [Google Scholar]
  • Hurwitz J, Dean FB, Kwong AD, Lee SH. The in vitro replication of DNA containing the SV40 origin. J Biol Chem. 1990 Oct 25;265(30):18043–18046. [PubMed] [Google Scholar]
  • Ishimi Y, Claude A, Bullock P, Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  • Johnston GC, Singer RA. Growth and the cell cycle of the yeast Saccharomyces cerevisiae. I. Slowing S phase or nuclear division decreases the G1 cell cycle period. Exp Cell Res. 1983 Nov;149(1):1–13. [PubMed] [Google Scholar]
  • Laskey RA, Fairman MP, Blow JJ. S phase of the cell cycle. Science. 1989 Nov 3;246(4930):609–614. [PubMed] [Google Scholar]
  • Longhese MP, Jovine L, Plevani P, Lucchini G. Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex. Genetics. 1993 Feb;133(2):183–191. [PMC free article] [PubMed] [Google Scholar]
  • Lucchini G, Muzi Falconi M, Pizzagalli A, Aguilera A, Klein HL, Plevani P. Nucleotide sequence and characterization of temperature-sensitive pol1 mutants of Saccharomyces cerevisiae. Gene. 1990 May 31;90(1):99–104. [PubMed] [Google Scholar]
  • Miyazawa H, Izumi M, Tada S, Takada R, Masutani M, Ui M, Hanaoka F. Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase alpha-primase complex and their gene expression during cell proliferation and the cell cycle. J Biol Chem. 1993 Apr 15;268(11):8111–8122. [PubMed] [Google Scholar]
  • Murakami Y, Hurwitz J. DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication. J Biol Chem. 1993 May 25;268(15):11018–11027. [PubMed] [Google Scholar]
  • Murakami Y, Wobbe CR, Weissbach L, Dean FB, Hurwitz J. Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1986 May;83(9):2869–2873. [PMC free article] [PubMed] [Google Scholar]
  • Murray AW. Creative blocks: cell-cycle checkpoints and feedback controls. Nature. 1992 Oct 15;359(6396):599–604. [PubMed] [Google Scholar]
  • Nasheuer HP, Moore A, Wahl AF, Wang TS. Cell cycle-dependent phosphorylation of human DNA polymerase alpha. J Biol Chem. 1991 Apr 25;266(12):7893–7903. [PubMed] [Google Scholar]
  • Nasmyth K. Control of the yeast cell cycle by the Cdc28 protein kinase. Curr Opin Cell Biol. 1993 Apr;5(2):166–179. [PubMed] [Google Scholar]
  • Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. [PubMed] [Google Scholar]
  • Panzeri L, Landonio L, Stotz A, Philippsen P. Role of conserved sequence elements in yeast centromere DNA. EMBO J. 1985 Jul;4(7):1867–1874. [PMC free article] [PubMed] [Google Scholar]
  • Pizzagalli A, Valsasnini P, Plevani P, Lucchini G. DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3772–3776. [PMC free article] [PubMed] [Google Scholar]
  • Plevani P, Foiani M, Muzi Falconi M, Pizzagalli A, Santocanale C, Francesconi S, Valsasnini P, Comedini A, Piatti S, Lucchini G. The yeast DNA polymerase-primase complex: genes and proteins. Biochim Biophys Acta. 1988 Dec 20;951(2-3):268–273. [PubMed] [Google Scholar]
  • Plevani P, Foiani M, Valsasnini P, Badaracco G, Cheriathundam E, Chang LM. Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex. J Biol Chem. 1985 Jun 10;260(11):7102–7107. [PubMed] [Google Scholar]
  • Plevani P, Francesconi S, Lucchini G. The nucleotide sequence of the PRI1 gene related to DNA primase in Saccharomyces cerevisiae. Nucleic Acids Res. 1987 Oct 12;15(19):7975–7989. [PMC free article] [PubMed] [Google Scholar]
  • Pringle JR. The use of conditional lethal cell cycle mutants for temporal and functional sequence mapping of cell cycle events. J Cell Physiol. 1978 Jun;95(3):393–405. [PubMed] [Google Scholar]
  • Pringle JR, Preston RA, Adams AE, Stearns T, Drubin DG, Haarer BK, Jones EW. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. [PubMed] [Google Scholar]
  • Reed SI. The role of p34 kinases in the G1 to S-phase transition. Annu Rev Cell Biol. 1992;8:529–561. [PubMed] [Google Scholar]
  • Roberts JM. Turning DNA replication on and off. Curr Opin Cell Biol. 1993 Apr;5(2):201–206. [PubMed] [Google Scholar]
  • Rowley A, Johnston GC, Butler B, Werner-Washburne M, Singer RA. Heat shock-mediated cell cycle blockage and G1 cyclin expression in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1034–1041. [PMC free article] [PubMed] [Google Scholar]
  • Santocanale C, Foiani M, Lucchini G, Plevani P. The isolated 48,000-dalton subunit of yeast DNA primase is sufficient for RNA primer synthesis. J Biol Chem. 1993 Jan 15;268(2):1343–1348. [PubMed] [Google Scholar]
  • Santocanale C, Locati F, Muzi Falconi M, Piseri A, Tseng BY, Lucchini G, Plevani P. Overproduction and functional analysis of DNA primase subunits from yeast and mouse. Gene. 1992 Apr 15;113(2):199–205. [PubMed] [Google Scholar]
  • Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PMC free article] [PubMed] [Google Scholar]
  • Slater ML. Effect of reversible inhibition of deoxyribonucleic acid synthesis on the yeast cell cycle. J Bacteriol. 1973 Jan;113(1):263–270. [PMC free article] [PubMed] [Google Scholar]
  • Tsurimoto T, Melendy T, Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. [PubMed] [Google Scholar]
  • Wang TS. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. [PubMed] [Google Scholar]
  • Weinert TA, Hartwell LH. Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics. 1993 May;134(1):63–80. [PMC free article] [PubMed] [Google Scholar]
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