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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Feb 1;91(3):868–872. doi: 10.1073/pnas.91.3.868

Molecular cloning and expression of the Saccharomyces cerevisiae RFC3 gene, an essential component of replication factor C.

X Li 1, P M Burgers 1
PMCID: PMC521413  PMID: 8302859

Abstract

Yeast replication factor C (RF-C) is a multi-polypeptide complex required for processive DNA replication by DNA polymerases delta and epsilon. The gene encoding the 40-kDa subunit of the Saccharomyces cerevisiae RF-C (RFC3) has been cloned. The RFC3 gene is required for yeast cell growth and has been mapped to the left arm of chromosome XIV. The deduced amino acid sequence of the RFC3 gene shows a high homology to the 36-, 37-, and 40-kDa subunits of human RF-C (also called activator 1), with the highest homology to the 36-kDa subunit. Among the conserved regions are the A motif of ATP binding proteins; the "DEAD box," common to DNA helicases and other ATPases; and the "RFC box," an approximately 15-amino acid domain virtually identical in the yeast and human RF-C subunits. Limited homology to the functional homologs of the Escherichia coli replication apparatus was also observed. The steady-state mRNA levels of RFC3 do not change significantly during the mitotic cell cycle of yeast. The intact form of the RFC3 gene product (Rfc3p) has been overproduced in E. coli and purified to homogeneity. Purified Rfc3p has an ATPase activity that is markedly stimulated by single-stranded DNA but not by double-stranded DNA or RNA.

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Selected References

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  1. Bauer G. A., Burgers P. M. Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res. 1990 Jan 25;18(2):261–265. doi: 10.1093/nar/18.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer G. A., Heller H. M., Burgers P. M. DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization. J Biol Chem. 1988 Jan 15;263(2):917–924. [PubMed] [Google Scholar]
  3. Boulet A., Simon M., Faye G., Bauer G. A., Burgers P. M. Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. EMBO J. 1989 Jun;8(6):1849–1854. doi: 10.1002/j.1460-2075.1989.tb03580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Burgers P. M., Bambara R. A., Campbell J. L., Chang L. M., Downey K. M., Hübscher U., Lee M. Y., Linn S. M., So A. G., Spadari S. Revised nomenclature for eukaryotic DNA polymerases. Eur J Biochem. 1990 Aug 17;191(3):617–618. doi: 10.1111/j.1432-1033.1990.tb19165.x. [DOI] [PubMed] [Google Scholar]
  6. Burgers P. M. Eukaryotic DNA polymerases alpha and delta: conserved properties and interactions, from yeast to mammalian cells. Prog Nucleic Acid Res Mol Biol. 1989;37:235–280. doi: 10.1016/s0079-6603(08)60700-x. [DOI] [PubMed] [Google Scholar]
  7. Burgers P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991 Nov 25;266(33):22698–22706. [PubMed] [Google Scholar]
  8. Chen M., Pan Z. Q., Hurwitz J. Sequence and expression in Escherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of HeLa cells. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2516–2520. doi: 10.1073/pnas.89.7.2516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen M., Pan Z. Q., Hurwitz J. Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5211–5215. doi: 10.1073/pnas.89.12.5211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Claassen L. A., Ahn B., Koo H. S., Grossman L. Construction of deletion mutants of the Escherichia coli UvrA protein and their purification from inclusion bodies. J Biol Chem. 1991 Jun 15;266(17):11380–11387. [PubMed] [Google Scholar]
  11. Fien K., Stillman B. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex. Mol Cell Biol. 1992 Jan;12(1):155–163. doi: 10.1128/mcb.12.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Impellizzeri K. J., Anderson B., Burgers P. M. The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair. J Bacteriol. 1991 Nov;173(21):6807–6810. doi: 10.1128/jb.173.21.6807-6810.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lee S. H., Hurwitz J. Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5672–5676. doi: 10.1073/pnas.87.15.5672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee S. H., Kwong A. D., Pan Z. Q., Hurwitz J. Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta. J Biol Chem. 1991 Jan 5;266(1):594–602. [PubMed] [Google Scholar]
  15. Lee S. H., Pan Z. Q., Kwong A. D., Burgers P. M., Hurwitz J. Synthesis of DNA by DNA polymerase epsilon in vitro. J Biol Chem. 1991 Nov 25;266(33):22707–22717. [PubMed] [Google Scholar]
  16. Lehman I. R., Kaguni L. S. DNA polymerase alpha. J Biol Chem. 1989 Mar 15;264(8):4265–4268. [PubMed] [Google Scholar]
  17. Li X., Yoder B. L., Burgers P. M. A Saccharomyces cerevisiae DNA helicase associated with replication factor C. J Biol Chem. 1992 Dec 15;267(35):25321–25327. [PubMed] [Google Scholar]
  18. Morrison A., Araki H., Clark A. B., Hamatake R. K., Sugino A. A third essential DNA polymerase in S. cerevisiae. Cell. 1990 Sep 21;62(6):1143–1151. doi: 10.1016/0092-8674(90)90391-q. [DOI] [PubMed] [Google Scholar]
  19. O'Donnell M., Onrust R., Dean F. B., Chen M., Hurwitz J. Homology in accessory proteins of replicative polymerases--E. coli to humans. Nucleic Acids Res. 1993 Jan 11;21(1):1–3. doi: 10.1093/nar/21.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ogas J., Andrews B. J., Herskowitz I. Transcriptional activation of CLN1, CLN2, and a putative new G1 cyclin (HCS26) by SWI4, a positive regulator of G1-specific transcription. Cell. 1991 Sep 6;66(5):1015–1026. doi: 10.1016/0092-8674(91)90445-5. [DOI] [PubMed] [Google Scholar]
  21. Pan Z. Q., Chen M., Hurwitz J. The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):6–10. doi: 10.1073/pnas.90.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  24. Simon M., Giot L., Faye G. The 3' to 5' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication. EMBO J. 1991 Aug;10(8):2165–2170. doi: 10.1002/j.1460-2075.1991.tb07751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sitney K. C., Budd M. E., Campbell J. L. DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene. Cell. 1989 Feb 24;56(4):599–605. doi: 10.1016/0092-8674(89)90582-5. [DOI] [PubMed] [Google Scholar]
  26. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tsurimoto T., Stillman B. Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1023–1027. doi: 10.1073/pnas.87.3.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsurimoto T., Stillman B. Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro. Mol Cell Biol. 1989 Feb;9(2):609–619. doi: 10.1128/mcb.9.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J Biol Chem. 1991 Jan 25;266(3):1950–1960. [PubMed] [Google Scholar]
  30. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J Biol Chem. 1991 Jan 25;266(3):1961–1968. [PubMed] [Google Scholar]
  31. Verma R., Smiley J., Andrews B., Campbell J. L. Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9479–9483. doi: 10.1073/pnas.89.20.9479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  33. Yoder B. L., Burgers P. M. Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity. J Biol Chem. 1991 Nov 25;266(33):22689–22697. [PubMed] [Google Scholar]
  34. Zhang J., Chung D. W., Tan C. K., Downey K. M., Davie E. W., So A. G. Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases. Biochemistry. 1991 Dec 24;30(51):11742–11750. doi: 10.1021/bi00115a002. [DOI] [PubMed] [Google Scholar]

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