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 T y 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 SUP#-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 T y insertion, but did not affect the rates of the other mutational classes detected. Relative to the wild-type parent, T y 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 T y 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 SUP#-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. P ROTEIN modification by attachment of ubiquitin has been implicated in the control of protein degradation and nonproteolytic modulation of protein structure and activity in eukaryotic cells (for reviews, see HERSHKO 1988; JENTSCH et a l . 1990; MONIA, ECKER and CROOKE 1990; JENTSCH, SEUFERT and HAUSER 1991). Ligation of ubiquitin to target proteins is a multistep process initiated by ATP-dependent binding of ubiquitin to the ubiquitin-activating enzyme (El). Next, the activated ubiquitin s transferred to ubiquitin-conjugating enzymes (E2s). Finally, the E2 enzymes catalyze the production of an isopeptide bond between ubiquitin and target proteins. Alternatively, prior to catalysis, the E2s may interact with ubiquitin-protein ligase (E3) which functions in substrate recognition. At least nine E2s have been identified in the yeast Saccharomyces cerevisiae and the corresponding genes have been isolated (JENTSCH, SEUFERT and HAUSER 1991; QIN et al . 1991). One of these (UBC2) is the previously known gene RAD6 (JENTSCH, MCGRATH ' To whom correspondence should be addressed. Genetics 130 285-294 (February, 1992) and VARSHAVSKY 1987). Consistent with an important role for ubiquitin conjugation in DNA metabolism, inactivation of RAD6 confers a highly pleiotropic phenotype. The reported properties of rad6 mutants include extreme sensitivity to a wide variety of DNAdamaging agents, increased spontaneous and induced mitotic recombination, enhanced spontaneous mutation rates, and deficiencies in post-replication repair, meiotic recombination, sporulation, and most types of induced mutagenesis (reviewed in HAYNES and KUNZ 1981; KUNZ and HAYNES 1981; LAWRENCE 1982; GAME 1983; FRIEDBERG 1988). These features may reflect loss of the ubiquitin-conjugating activity since directed mutation of the single cysteine residue present in the RAD6 E2 eliminates the capacity of the enzyme to bind ubiquitin and results in the same defects as deletion of RAD6 (SUNG, PRAKASH and PRAKASH 1990). It remains possible, however, that another activity involving this cysteine residue accounts for the rad6 phenotype. In vitro, the core histones H2A, H2B and H3 are targets for the RAD6 enzyme UENTSCH, MCGRATH