Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2001 Sep;159(1):47–64. doi: 10.1093/genetics/159.1.47

In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.

Y I Pavlov 1, P V Shcherbakova 1, T A Kunkel 1
PMCID: PMC1461793  PMID: 11560886

Abstract

Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases alpha, delta, epsilon, and zeta and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Pol delta or Pol epsilon are inviable. Strains with same substitution in Rev3, the catalytic subunit of Pol zeta, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Pol alpha (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Pol alpha, -delta, or -epsilon are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Pol alpha mutant has no obvious phenotype. The Pol epsilon (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Pol delta mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Pol alpha, -delta, and -epsilon mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.

Full Text

The Full Text of this article is available as a PDF (265.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Astatke M., Ng K., Grindley N. D., Joyce C. M. A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3402–3407. doi: 10.1073/pnas.95.7.3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell J. B., Eckert K. A., Joyce C. M., Kunkel T. A. Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain. J Biol Chem. 1997 Mar 14;272(11):7345–7351. doi: 10.1074/jbc.272.11.7345. [DOI] [PubMed] [Google Scholar]
  3. Bemark M., Khamlichi A. A., Davies S. L., Neuberger M. S. Disruption of mouse polymerase zeta (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro. Curr Biol. 2000 Oct 5;10(19):1213–1216. doi: 10.1016/s0960-9822(00)00724-7. [DOI] [PubMed] [Google Scholar]
  4. Budd M. E., Campbell J. L. DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jan;13(1):496–505. doi: 10.1128/mcb.13.1.496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Budd M., Campbell J. L. Temperature-sensitive mutations in the yeast DNA polymerase I gene. Proc Natl Acad Sci U S A. 1987 May;84(9):2838–2842. doi: 10.1073/pnas.84.9.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carroll S. S., Cowart M., Benkovic S. J. A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity. Biochemistry. 1991 Jan 22;30(3):804–813. doi: 10.1021/bi00217a034. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  8. Chen C., Merrill B. J., Lau P. J., Holm C., Kolodner R. D. Saccharomyces cerevisiae pol30 (proliferating cell nuclear antigen) mutations impair replication fidelity and mismatch repair. Mol Cell Biol. 1999 Nov;19(11):7801–7815. doi: 10.1128/mcb.19.11.7801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen C., Umezu K., Kolodner R. D. Chromosomal rearrangements occur in S. cerevisiae rfa1 mutator mutants due to mutagenic lesions processed by double-strand-break repair. Mol Cell. 1998 Jul;2(1):9–22. doi: 10.1016/s1097-2765(00)80109-4. [DOI] [PubMed] [Google Scholar]
  10. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  11. Dong Q., Copeland W. C., Wang T. S. Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis. J Biol Chem. 1993 Nov 15;268(32):24163–24174. [PubMed] [Google Scholar]
  12. Donlin M. J., Johnson K. A. Mutants affecting nucleotide recognition by T7 DNA polymerase. Biochemistry. 1994 Dec 13;33(49):14908–14917. doi: 10.1021/bi00253a030. [DOI] [PubMed] [Google Scholar]
  13. Esposito G., Godindagger I., Klein U., Yaspo M. L., Cumano A., Rajewsky K. Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in mice results in early embryonic lethality. Curr Biol. 2000 Oct 5;10(19):1221–1224. doi: 10.1016/s0960-9822(00)00726-0. [DOI] [PubMed] [Google Scholar]
  14. Friedberg E. C., Feaver W. J., Gerlach V. L. The many faces of DNA polymerases: strategies for mutagenesis and for mutational avoidance. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5681–5683. doi: 10.1073/pnas.120152397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Friedberg E. C., Gerlach V. L. Novel DNA polymerases offer clues to the molecular basis of mutagenesis. Cell. 1999 Aug 20;98(4):413–416. doi: 10.1016/s0092-8674(00)81970-4. [DOI] [PubMed] [Google Scholar]
  16. Fujii S., Akiyama M., Aoki K., Sugaya Y., Higuchi K., Hiraoka M., Miki Y., Saitoh N., Yoshiyama K., Ihara K. DNA replication errors produced by the replicative apparatus of Escherichia coli. J Mol Biol. 1999 Jun 18;289(4):835–850. doi: 10.1006/jmbi.1999.2802. [DOI] [PubMed] [Google Scholar]
  17. Goldstein A. L., McCusker J. H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast. 1999 Oct;15(14):1541–1553. doi: 10.1002/(SICI)1097-0061(199910)15:14<1541::AID-YEA476>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  18. Goodman M. F., Tippin B. The expanding polymerase universe. Nat Rev Mol Cell Biol. 2000 Nov;1(2):101–109. doi: 10.1038/35040051. [DOI] [PubMed] [Google Scholar]
  19. Harfe B. D., Jinks-Robertson S. DNA polymerase zeta introduces multiple mutations when bypassing spontaneous DNA damage in Saccharomyces cerevisiae. Mol Cell. 2000 Dec;6(6):1491–1499. doi: 10.1016/s1097-2765(00)00145-3. [DOI] [PubMed] [Google Scholar]
  20. Holbeck S. L., Strathern J. N. A role for REV3 in mutagenesis during double-strand break repair in Saccharomyces cerevisiae. Genetics. 1997 Nov;147(3):1017–1024. doi: 10.1093/genetics/147.3.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hopfner K. P., Eichinger A., Engh R. A., Laue F., Ankenbauer W., Huber R., Angerer B. Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3600–3605. doi: 10.1073/pnas.96.7.3600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnson R. E., Washington M. T., Haracska L., Prakash S., Prakash L. Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. Nature. 2000 Aug 31;406(6799):1015–1019. doi: 10.1038/35023030. [DOI] [PubMed] [Google Scholar]
  23. Kesti T., Flick K., Keränen S., Syväoja J. E., Wittenberg C. DNA polymerase epsilon catalytic domains are dispensable for DNA replication, DNA repair, and cell viability. Mol Cell. 1999 May;3(5):679–685. doi: 10.1016/s1097-2765(00)80361-5. [DOI] [PubMed] [Google Scholar]
  24. Kirchner J. M., Tran H., Resnick M. A. A DNA polymerase epsilon mutant that specifically causes +1 frameshift mutations within homonucleotide runs in yeast. Genetics. 2000 Aug;155(4):1623–1632. doi: 10.1093/genetics/155.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kokoska R. J., Stefanovic L., Tran H. T., Resnick M. A., Gordenin D. A., Petes T. D. Destabilization of yeast micro- and minisatellite DNA sequences by mutations affecting a nuclease involved in Okazaki fragment processing (rad27) and DNA polymerase delta (pol3-t). Mol Cell Biol. 1998 May;18(5):2779–2788. doi: 10.1128/mcb.18.5.2779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lawrence C. W., Hinkle D. C. DNA polymerase zeta and the control of DNA damage induced mutagenesis in eukaryotes. Cancer Surv. 1996;28:21–31. [PubMed] [Google Scholar]
  27. Lühr B., Scheller J., Meyer P., Kramer W. Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae. Mol Gen Genet. 1998 Feb;257(3):362–367. doi: 10.1007/s004380050658. [DOI] [PubMed] [Google Scholar]
  28. Marsischky G. T., Filosi N., Kane M. F., Kolodner R. Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair. Genes Dev. 1996 Feb 15;10(4):407–420. doi: 10.1101/gad.10.4.407. [DOI] [PubMed] [Google Scholar]
  29. Masutani C., Araki M., Yamada A., Kusumoto R., Nogimori T., Maekawa T., Iwai S., Hanaoka F. Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity. EMBO J. 1999 Jun 15;18(12):3491–3501. doi: 10.1093/emboj/18.12.3491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McAlear M. A., Tuffo K. M., Holm C. The large subunit of replication factor C (Rfc1p/Cdc44p) is required for DNA replication and DNA repair in Saccharomyces cerevisiae. Genetics. 1996 Jan;142(1):65–78. doi: 10.1093/genetics/142.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McDonald J. P., Levine A. S., Woodgate R. The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism. Genetics. 1997 Dec;147(4):1557–1568. doi: 10.1093/genetics/147.4.1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Minnick D. T., Bebenek K., Osheroff W. P., Turner R. M., Jr, Astatke M., Liu L., Kunkel T. A., Joyce C. M. Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment). J Biol Chem. 1999 Jan 29;274(5):3067–3075. doi: 10.1074/jbc.274.5.3067. [DOI] [PubMed] [Google Scholar]
  33. Morrison A., Bell J. B., Kunkel T. A., Sugino A. Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9473–9477. doi: 10.1073/pnas.88.21.9473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Morrison A., Christensen R. B., Alley J., Beck A. K., Bernstine E. G., Lemontt J. F., Lawrence C. W. REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase. J Bacteriol. 1989 Oct;171(10):5659–5667. doi: 10.1128/jb.171.10.5659-5667.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Morrison A., Johnson A. L., Johnston L. H., Sugino A. Pathway correcting DNA replication errors in Saccharomyces cerevisiae. EMBO J. 1993 Apr;12(4):1467–1473. doi: 10.1002/j.1460-2075.1993.tb05790.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Morrison A., Sugino A. Roles of POL3, POL2 and PMS1 genes in maintaining accurate DNA replication. Chromosoma. 1992;102(1 Suppl):S147–S149. doi: 10.1007/BF02451799. [DOI] [PubMed] [Google Scholar]
  37. Morrison A., Sugino A. The 3'-->5' exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae. Mol Gen Genet. 1994 Feb;242(3):289–296. doi: 10.1007/BF00280418. [DOI] [PubMed] [Google Scholar]
  38. Mortimer R. K., Contopoulou R., Schild D. Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5778–5782. doi: 10.1073/pnas.78.9.5778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nakano Y., Yoshida Y., Yamashita Y., Koga T. Construction of a series of pACYC-derived plasmid vectors. Gene. 1995 Aug 30;162(1):157–158. doi: 10.1016/0378-1119(95)00320-6. [DOI] [PubMed] [Google Scholar]
  41. Perrino F. W., Loeb L. A. Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha. Biochemistry. 1990 Jun 5;29(22):5226–5231. doi: 10.1021/bi00474a002. [DOI] [PubMed] [Google Scholar]
  42. Polesky A. H., Dahlberg M. E., Benkovic S. J., Grindley N. D., Joyce C. M. Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli. J Biol Chem. 1992 Apr 25;267(12):8417–8428. [PubMed] [Google Scholar]
  43. Reha-Krantz L. J., Nonay R. L. Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity. Isolation of new antimutator and mutator DNA polymerases. J Biol Chem. 1994 Feb 25;269(8):5635–5643. [PubMed] [Google Scholar]
  44. Shcherbakova P. V., Kunkel T. A. Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations. Mol Cell Biol. 1999 Apr;19(4):3177–3183. doi: 10.1128/mcb.19.4.3177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shcherbakova P. V., Noskov V. N., Pshenichnov M. R., Pavlov Y. I. Base analog 6-N-hydroxylaminopurine mutagenesis in the yeast Saccharomyces cerevisiae is controlled by replicative DNA polymerases. Mutat Res. 1996 Jul 10;369(1-2):33–44. doi: 10.1016/s0165-1218(96)90045-2. [DOI] [PubMed] [Google Scholar]
  46. Shcherbakova P. V., Pavlov Y. I. 3'-->5' exonucleases of DNA polymerases epsilon and delta correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae. Genetics. 1996 Mar;142(3):717–726. doi: 10.1093/genetics/142.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Thomas B. J., Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989 Feb 24;56(4):619–630. doi: 10.1016/0092-8674(89)90584-9. [DOI] [PubMed] [Google Scholar]
  48. Tran H. T., Degtyareva N. P., Gordenin D. A., Resnick M. A. Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast. Genetics. 1999 May;152(1):47–59. doi: 10.1093/genetics/152.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tran H. T., Degtyareva N. P., Koloteva N. N., Sugino A., Masumoto H., Gordenin D. A., Resnick M. A. Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes. Mol Cell Biol. 1995 Oct;15(10):5607–5617. doi: 10.1128/mcb.15.10.5607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tran H., Degtyareva N., Gordenin D., Resnick M. A. Altered replication and inverted repeats induce mismatch repair-independent recombination between highly diverged DNAs in yeast. Mol Cell Biol. 1997 Feb;17(2):1027–1036. doi: 10.1128/mcb.17.2.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
  52. Waga S., Stillman B. The DNA replication fork in eukaryotic cells. Annu Rev Biochem. 1998;67:721–751. doi: 10.1146/annurev.biochem.67.1.721. [DOI] [PubMed] [Google Scholar]
  53. Wang J., Sattar A. K., Wang C. C., Karam J. D., Konigsberg W. H., Steitz T. A. Crystal structure of a pol alpha family replication DNA polymerase from bacteriophage RB69. Cell. 1997 Jun 27;89(7):1087–1099. doi: 10.1016/s0092-8674(00)80296-2. [DOI] [PubMed] [Google Scholar]
  54. Wilson T. E., Lieber M. R. Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway. J Biol Chem. 1999 Aug 13;274(33):23599–23609. doi: 10.1074/jbc.274.33.23599. [DOI] [PubMed] [Google Scholar]
  55. Wittschieben J., Shivji M. K., Lalani E., Jacobs M. A., Marini F., Gearhart P. J., Rosewell I., Stamp G., Wood R. D. Disruption of the developmentally regulated Rev3l gene causes embryonic lethality. Curr Biol. 2000 Oct 5;10(19):1217–1220. doi: 10.1016/s0960-9822(00)00725-9. [DOI] [PubMed] [Google Scholar]
  56. Yang G., Lin T., Karam J., Konigsberg W. H. Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation. Biochemistry. 1999 Jun 22;38(25):8094–8101. doi: 10.1021/bi990653w. [DOI] [PubMed] [Google Scholar]
  57. Zhao Y., Jeruzalmi D., Moarefi I., Leighton L., Lasken R., Kuriyan J. Crystal structure of an archaebacterial DNA polymerase. Structure. 1999 Oct 15;7(10):1189–1199. doi: 10.1016/s0969-2126(00)80053-2. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES