Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae.
We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.
Dornfeld
K J
KJ
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Livingston
D M
DM
D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008938:Mitosis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D004271:DNA, Fungal; D008741:Methyl Methanesulfonate