Multiple recombination pathways for sister chromatid exchange in Saccharomyces cerevisiae: role of RAD1 and the RAD52 epistasis group genes

Abstract

Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those directly initiated by double-strand breaks (DSBs), such as gap repair and break-induced replication (BIR), and those initiated when DNA polymerases stall, such as template switching. To elucidate SCE recombination mechanisms, we determined whether spontaneous and DNA damage-associated SCE requires specific genes within the RAD52 and RAD3 epistasis groups in Saccharomyces cerevisiae strains containing two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs. SCE frequencies were measured after cells were exposed to UV, X-rays, 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS), or when an HO endonuclease-induced DSB was introduced at his3-Delta3'::HOcs. Our data indicate that genes involved in gap repair, such as RAD55, RAD57 and RAD54, are required for DNA damage-associated SCE but not for spontaneous SCE. RAD50 and RAD59, genes required for BIR, are required for X-ray-associated SCE but not for SCE stimulated by HO-induced DSBs. In comparison with wild type, rates of spontaneous SCE are 10-fold lower in rad51 rad1 but not in either rad51 rad50 or rad51 rad59 double mutants. We propose that gap repair mechanisms are important in DNA damage-associated recombination, whereas alternative pathways, including a template switch pathway, play a role in spontaneous SCE.

A Mutant Allele of the Transcription Factor IIH Helicase Gene, RAD3, Promotes Loss of Heterozygosity in Response to a DNA Replication Defect in Saccharomyces cerevisiae

“…Interestingly, the presence of a homolog failed to stimulate direct-repeat recombination in the rad3-102 rad27-null double-mutant diploids beyond that observed in the rad27-null single-mutant haploids, and these rates were not significantly different from those observed in rad27-null and rad3-102 rad27-null haploids (Table 3). This is consistent with fork collapse leading to the formation of single-ended DSBs that are thought to be ideal substrates for interhomolog recombination by BIR (McEachern and Haber 2006), but not, perhaps, direct-repeat recombination, which is thought to occur by single-strand annealing (Lin et al 1990;Ivanov et al 1996;Dong and Fasullo 2003;Davis and Symington 2004).…”

“…Unequal sister-chromatid and direct-repeat recombination are unaffected by the rad3-102 allele in the rad3-102 rad27-null double mutants: Template switching with the sister chromatid is another response to replication lesions (Dong and Fasullo 2003). Consequently, we investigated the epistatic relationship between the rad3-102 and rad27-null mutations with respect to USCR using an assay developed by Fasullo and Davis (1987).…”

Role of the Saccharomyces cerevisiae Rad51 Paralogs in Sister Chromatid Recombination

“…DSBs have been detected at some inverted repeats in yeast and may form at low frequency within the ade2-inverted repeat (Lobachev et al 2002;Lemoine et al 2005). In an assay to measure spontaneous unequal sister chromatid exchange, rad51, rad55, and rad57 mutants were found to have the same rate as wild type (Dong and Fasullo 2003). The failure to detect Rad51-dependent events in this system could be due to the selection for exchange events, the short homology between the repeats (0.3 kb), or use of an alternate postreplication repair pathway, such as template switching, to generate recombinants.…”

Activation of the DNA Damage Checkpoint in Yeast Lacking the Histone Chaperone Anti-Silencing Function 1

“…We used an assay to measure SCE, a recombination event that is initiated in S phase (2,9). Wild-type and asf1 mutant strains containing a 5Ј fragment of HIS3 next to a 3Ј fragment of HIS3 were used to quantitate spontaneous SCE by the formation of a functional HIS3 gene, as described previously (12).…”