Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non-homologous repair paths

Abstract

Eukaryotes have acquired many mechanisms to repair DNA double-strand breaks (DSBs) [1]. In the yeast Saccharomyces cerevisiae, this damage can be repaired either by homologous recombination, which depends on the Rad52 protein, or by non-homologous end-joining (NHEJ), which depends on the proteins yKu70 and yKu80 [2] [3]. How do cells choose which repair pathway to use? Deletions of the SIR2, SIR3 and SIR4 genes - which are involved in transcriptional silencing at telomeres and HM mating-type loci (HMLalpha and HMRa) in yeast [4] - have been reported to reduce NHEJ as severely as deletions of genes encoding Ku proteins [5]. Here, we report that the effect of deleting SIR genes is largely attributable to derepression of silent mating-type genes, although Sir proteins do play a minor role in end-joining. When DSBs were made on chromosomes in haploid cells that retain their mating type, sir Delta mutants reduced the frequency of NHEJ by twofold or threefold, although plasmid end-joining was not affected. In diploid cells, sir mutants showed a twofold reduction in the frequency of NHEJ in two assays. Mating type also regulated the efficiency of DSB-induced homologous recombination. In MATa/MATalpha diploid cells, a DSB induced by HO endonuclease was repaired 98% of the time by gene conversion with the homologous chromosome, whereas in diploid cells with an alpha mating type (matDelta/MATalpha) repair succeeded only 82% of the time. Mating-type regulation of genes specific to haploid or diploid cells plays a key role in determining which pathways are used to repair DSBs.

Differential Usage of Alternative Pathways of Double-Strand Break Repair in Drosophila

“…The prevalence of different break repair pathways has been tested in brewer's and fission yeast, as well as in Arabidopsis, chicken, and mammalian cells (Lee et al 1999;Wang et al 2001;Wilson 2002;Orel et al 2003;Prudden et al 2003;Stark et al 2004;Elliott et al 2005;Nakanishi et al 2005). In these experiments as in ours, whenever SSA was available it was relatively efficient.…”

Mismatch Tolerance by DNA Polymerase Pol4 in the Course of Nonhomologous End Joining in Saccharomyces cerevisiae

“…between repair of transformed DNA ends and repair of a break generated in vivo: the absolute repair efficiency is lower and the error rate higher with the plasmid transformation assay (Lee et al 1999;Frank-Vaillant and Marcand 2002;Karathanasis and Wilson 2002).…”

“…The ligation step is performed by an ATP-dependent DNA ligase committed to this pathway, Lig4 and its associated factor Lif1 in Saccharomyces cerevisiae. Repair by NHEJ of DSBs whose ends are perfectly cohesive is essentially a ligation and is a very efficient and accurate process (Lee et al 1999;Frank-Vaillant and Marcand 2002). NHEJ may also attempt to restore the original sequence at a DSB whose ends are uncohesive due to damaged bases.…”

Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair

“…It was shown that cells lacking these factors exhibit a defect in NHEJ, and furthermore, chromatin immunoprecipitation (ChIP) studies showed that these factors relocalize from telomeres to the site of DNA DSBs (18)(19)(20)(21). Despite this localization, it is now clear that the defect of cells lacking these factors in NHEJ is mainly indirect because deletion of the SIR genes leads to pseudodiploidy in yeast as a result of loss of silencing at the silent mating type loci (22,23). This results in expression of the a͞␣ transcriptional regulator that suppresses a number of haploid-specific genes, including NEJ1, which encodes for a protein required for NHEJ (24,25).…”