The roles of REV3 and RAD57 in double-strand-break-repair-induced mutagenesis of Saccharomyces cerevisiae.
The DNA synthesis associated with recombinational repair of chromosomal double-strand breaks (DSBs) has a lower fidelity than normal replicative DNA synthesis. Here, we use an inverted-repeat substrate to monitor the fidelity of repair of a site-specific DSB. DSB induction made by the HO endonuclease stimulates recombination >5000-fold and is associated with a >1000-fold increase in mutagenesis of an adjacent gene. We demonstrate that most break-repair-induced mutations (BRIMs) are point mutations and have a higher proportion of frameshifts than do spontaneous mutations of the same substrate. Although the REV3 translesion DNA polymerase is not required for recombination, it introduces approximately 75% of the BRIMs and approximately 90% of the base substitution mutations. Recombinational repair of the DSB is strongly dependent upon genes of the RAD52 epistasis group; however, the residual recombinants present in rad57 mutants are associated with a 5- to 20-fold increase in BRIMs. The spectrum of mutations in rad57 mutants is similar to that seen in the wild-type strain and is similarly affected by REV3. We also find that REV3 is required for the repair of MMS-induced lesions when recombinational repair is compromised. Our data suggest that Rad55p/Rad57p help limit the generation of substrates that require pol zeta during recombination.
Rattray
Alison J
AJ
Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Shafer
Brenda K
BK
McGill
Carolyn B
CB
Strathern
Jeffrey N
JN
D000251:Adenosine Triphosphatases; D015139:Blotting, Southern; D004260:DNA Repair / Q000502:physiology; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000502:physiology*; D005656:Fungal Proteins / Q000502:physiology*; D009154:Mutation / Q000502:physiology*; D017354:Point Mutation; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D004259:DNA-Directed DNA Polymerase; C092602:REV3 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C071451:RAD57 protein, S cerevisiae; D045643:DNA Repair Enzymes