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.
N1 - Funding Information:
We thank Marilyn Diaz and Stephanie Nick McElhinny for thoughtful suggestions on this manuscript. We thank the NIEHS DNA sequencing core for their assistance. This work was supported in part by the Intramural Research Program of the NIH, NIEHS (to TAK) and in part by the Japan Atomic Energy Agency (to ANS).
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
C2 - 17715002
AN - SCOPUS:35948952789
VL - 6
SP - 1829
EP - 1838
JO - DNA Repair
JF - DNA Repair
SN - 1568-7864
IS - 12
ER -