Mutator alleles of yeast DNA polymerase zeta.
The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta 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 zeta 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 zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. 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 zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain 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 zeta functions in mammals, where REV3 deletion is lethal.
Sakamoto
Ayako N
AN
Research Group for Gene Resources, Department of Ion-Beam-Applied Biology, Japan Atomic Energy Agency, Watanuki-machi 1233, Takasaki, Gunma 370-1292, Japan.
Stone
Jana E
JE
Kissling
Grace E
GE
McCulloch
Scott D
SD
Pavlov
Youri I
YI
Kunkel
Thomas A
TA
D000483:Alleles; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014466:Ultraviolet Rays
C425929:DNA polymerase zeta; D004259:DNA-Directed DNA Polymerase
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