Mutations in RAD6, a yeast gene encoding a ubiquitin-conjugating enzyme, stimulate retrotransposition.

Abstract

The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CANI and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.Retrotransposons such as Ty are common in eucaryotes. While they are not infectious, they otherwise resemble retroviruses in their structural organization and replication in viruslike particles via an RNA intermediate (for a review, see reference 1). The finding that two-to fourfold increases in the transcription of Ty message caused disproportionately large increases in Ty transposition led to speculation that the cell may exert controls on transposition at a posttranscriptional level (7). Possibly the extra Ty mRNA leads to a complete saturation of this hypothetical posttranscriptional regulatory system. Other evidence that the rate of Ty transposition is not simply proportional to the Ty message level comes from the observation that growth at a low temperature (17 versus 30°C) causes a 50-to 100-fold increase in the rate of Ty transposition without changing the level of Ty mRNA (24,25). One explanation of this observation is that the reverse transcriptase activity is optimal at low temperatures (10).Mutations in the Saccharomyces cerevisiae RAD6 gene may result in several phenotypes, including extreme sensitivity to DNA-damaging agents, lack of induced mutagenesis, defective sporulation, increased mitotic recombination, and an increased rate of spontaneous mutation (for a review, see reference 13). RAD6 encodes an E22ok enzyme which polyubiquitinates histones H2A and H2B in vitro (16, 35) and which may influence chromatin structure (16,29,35). Here we show that among the spontaneous mutations stimulated by rad6 are mutations due to the transposition of Ty elements. Since the yeast RAD6 protein may be analogous to a mammalian 20-kilodalton E2 ubiquitin-conjugating enzyme (16,26), these studies may be relevant to studies of the cellular mechanisms used to control the movements of retrotransposons and retroviruses in other eucaryotes. LP2752-4B and LP2752-4Brad6A and plasmid pR67 were kindly provided by Louise Prakash. N-152, also from Louise Prakash, is a rad6-3 mutant (28) and was derived from B-635, a cycl-115 mutant derived from D311-3A (a Iys2 hisi trp2), both kindly provided by Fred Sherman.Mutant isolation. A large number of independent colonies from each strain were used to inoculate individual slants of complete medium, YPD (32), which were grown at 30°C. Cells suspended from these slants were plated either to synthetic complete medium lacking arginine and containing 60 ,ug of canavanine per ml (-Arg +Can) (32) or to medium containin...

Yeast Ty1 Retrotransposition Is Stimulated by a Synergistic Interaction between Mutations in Chromatin Assembly Factor I and Histone Regulatory Proteins

“…We previously reported that a deletion of RAD6 dramatically increases the rate of transposition of Ty1 into the CAN1 gene (54). This is in contrast to our finding, presented here, that deletions of RAD6 cause only a marginal increase in the transposition of Ty1 into the promoterless his3 target.…”

“…Mutations in RAD6 increase the overall rate of Ty1 transposition into CAN1 and SUP4 without causing an increase in the level of total Ty1 RNA (8,31,54). However, when genetically marked Ty1 elements were used, it was determined that the deletion of RAD6 affected the transposition of some, but not other, elements, and these effects were at the transcriptional level (8).…”

The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme.

“…The conspicuous and pleiotropic phenotype of ubc2(rad6) null mutations (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) stands in contrast to the relatively mild phenotype of ubrl null mutations, which also inactivate the N-end rule pathway (11). Thus, unlike N-recognin, whose known functions are confined to the N-end rule pathway (11), the UBC2 enzyme is an essential component not only of this pathway but at least of one other Ub-dependent pathway as well.…”

“…Two of the yeast UBC enzymes have been identified as the products of the previously known genes RAD6 (renamed UBC2) (22), which participates in DNA repair, induced mutagenesis, sporulation (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), and suppression of retrotransposition (34), and CDC34 (renamed UBC3), which is required for the transition from G1 to S phase of the cell cycle (35). Two of the other yeast UBC enzymes, UBC4 and UBC5, have been shown to be required for most of the Ub-dependent protein degradation in this organism (18,36).…”

A Novel Function of the DNA Repair Gene rhp6 in Mating-Type Silencing by Chromatin Remodeling in Fission Yeast

“…Though rhp6 and its homologs in other species have been known for several years, its actual biochemical function has not been clear. Earlier reports have demonstrated elevation and randomization of Ty transposition in the vicinity of certain genes in rad6 Ϫ mutants in S. cerevisiae (37,47), suggesting that Rad6 may affect the chromatin structure of the genes so as to make them better targets for Ty transposi- a Quantitation by densitometry of the autoradiograph shown in Fig. 8B.…”