Elimination of the yeast RAD6 ubiquitin conjugase enhances base-pair transitions and G.C----T.A transversions as well as transposition of the Ty element: implications for the control of spontaneous mutation.
The RAD6 gene of the yeast Saccharomyces cerevisiae encodes an enzyme that conjugates ubiquitin to other proteins. Defects in RAD6 confer a mutator phenotype due, in part, to an increased rate of transposition of the yeast Ty element. To further delineate the role of protein ubiquitination in the control of spontaneous mutagenesis in yeast, we have characterized 202 mutations that arose spontaneously in the SUP4-o gene carried on a centromere vector in a RAD6 deletion strain. The resulting mutational spectrum was compared to that for 354 spontaneous SUP4-o mutations isolated in the isogenic wild-type parent. This comparison revealed that the rad6 mutator enhanced the rate of single base-pair substitution, as well as Ty insertion, but did not affect the rates of the other mutational classes detected. Relative to the wild-type parent, Ty inserted at considerably more SUP4-o positions in the rad6 strain with a significantly smaller fraction detected at a transposition hotspot. These findings suggest that, in addition to the rate of transposition, protein ubiquitination might influence the target site specificity of Ty insertion. The increase in the substitution rate accounted for approximately 90% of the rad6 mutator effect but only the two transitions and the G. C----T.A transversion were enhanced. Analysis of the distribution of these events within SUP4-o suggested that the site specificity of the substitutions was influenced by DNA sequence context. Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad6 mutator did not reduce the efficiency of correcting mismatches that could give rise to the transitions or transversion nor did it bias restoration of the mismatches to the incorrect base-pairs. These results are discussed in relation to possible mechanisms that might link ubiquitination of proteins to spontaneous mutation rates.
Kang
X L
XL
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
Yadao
F
F
Gietz
R D
RD
Kunz
B A
BA
D001482:Base Composition; D001483:Base Sequence; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D008025:Ligases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases