Mutator alleles of yeast DNA polymerase ζ

Ayako N. Sakamoto, Jana E. Stone, Grace E. Kissling, Scott D. McCulloch, Youri I Pavlov, Thomas A. Kunkel

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol ζ), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol ζ mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol ζ active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol ζ catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol η was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Δ background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Δ rev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol ζ. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol ζ in vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol ζ in vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol ζ functions in mammals, where REV3 deletion is lethal.

Original languageEnglish (US)
Pages (from-to)1829-1838
Number of pages10
JournalDNA Repair
Volume6
Issue number12
DOIs
StatePublished - Dec 1 2007

Fingerprint

DNA-Directed DNA Polymerase
Yeast
Yeasts
Alleles
Catalytic Domain
Mutagenesis
Mutation
DNA
Catalytic DNA
Amino Acids
Mammals
Guanine
Mutation Rate
Catalyst activity
Repair
Substitution reactions
Genes
Irradiation

Keywords

  • Mutagenesis
  • Pol ζ
  • Translesion synthesis
  • Yeast

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Sakamoto, A. N., Stone, J. E., Kissling, G. E., McCulloch, S. D., Pavlov, Y. I., & Kunkel, T. A. (2007). Mutator alleles of yeast DNA polymerase ζ. DNA Repair, 6(12), 1829-1838. https://doi.org/10.1016/j.dnarep.2007.07.002

Mutator alleles of yeast DNA polymerase ζ. / Sakamoto, Ayako N.; Stone, Jana E.; Kissling, Grace E.; McCulloch, Scott D.; Pavlov, Youri I; Kunkel, Thomas A.

In: DNA Repair, Vol. 6, No. 12, 01.12.2007, p. 1829-1838.

Research output: Contribution to journalArticle

Sakamoto, AN, Stone, JE, Kissling, GE, McCulloch, SD, Pavlov, YI & Kunkel, TA 2007, 'Mutator alleles of yeast DNA polymerase ζ', DNA Repair, vol. 6, no. 12, pp. 1829-1838. https://doi.org/10.1016/j.dnarep.2007.07.002
Sakamoto AN, Stone JE, Kissling GE, McCulloch SD, Pavlov YI, Kunkel TA. Mutator alleles of yeast DNA polymerase ζ. DNA Repair. 2007 Dec 1;6(12):1829-1838. https://doi.org/10.1016/j.dnarep.2007.07.002
Sakamoto, Ayako N. ; Stone, Jana E. ; Kissling, Grace E. ; McCulloch, Scott D. ; Pavlov, Youri I ; Kunkel, Thomas A. / Mutator alleles of yeast DNA polymerase ζ. In: DNA Repair. 2007 ; Vol. 6, No. 12. pp. 1829-1838.
@article{2587404b044c4178b755841886e39857,
title = "Mutator alleles of yeast DNA polymerase ζ",
abstract = "The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol ζ), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol ζ mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol ζ active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol ζ catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol η was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Δ background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Δ rev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol ζ. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol ζ in vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol ζ in vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol ζ functions in mammals, where REV3 deletion is lethal.",
keywords = "Mutagenesis, Pol ζ, Translesion synthesis, Yeast",
author = "Sakamoto, {Ayako N.} and Stone, {Jana E.} and Kissling, {Grace E.} and McCulloch, {Scott D.} and Pavlov, {Youri I} and Kunkel, {Thomas A.}",
year = "2007",
month = "12",
day = "1",
doi = "10.1016/j.dnarep.2007.07.002",
language = "English (US)",
volume = "6",
pages = "1829--1838",
journal = "DNA Repair",
issn = "1568-7864",
publisher = "Elsevier",
number = "12",

}

TY - JOUR

T1 - Mutator alleles of yeast DNA polymerase ζ

AU - Sakamoto, Ayako N.

AU - Stone, Jana E.

AU - Kissling, Grace E.

AU - McCulloch, Scott D.

AU - Pavlov, Youri I

AU - Kunkel, Thomas A.

PY - 2007/12/1

Y1 - 2007/12/1

N2 - The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol ζ), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol ζ mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol ζ active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol ζ catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol η was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Δ background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Δ rev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol ζ. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol ζ in vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol ζ in vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol ζ functions in mammals, where REV3 deletion is lethal.

AB - The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol ζ), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol ζ mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol ζ active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol ζ catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol η was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Δ background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Δ rev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol ζ. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol ζ in vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol ζ in vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol ζ functions in mammals, where REV3 deletion is lethal.

KW - Mutagenesis

KW - Pol ζ

KW - Translesion synthesis

KW - Yeast

UR - http://www.scopus.com/inward/record.url?scp=35948952789&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=35948952789&partnerID=8YFLogxK

U2 - 10.1016/j.dnarep.2007.07.002

DO - 10.1016/j.dnarep.2007.07.002

M3 - Article

VL - 6

SP - 1829

EP - 1838

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

IS - 12

ER -