The Essential Helicase Gene RAD3 Suppresses Short-Sequence Recombination in Saccharomyces cerevisiae

Abstract

We have isolated an allele of the essential DNA repair and transcription gene RAD3 that relaxes the restriction against recombination between short DNA sequences in Saccharomyces cerevisiae. Double-strand break repair and gene replacement events requiring recombination between short identical or mismatched sequences were stimulated in the rad3-G595R mutant cells. We also observed an increase in the physical stability of double-strand breaks in the rad3-G595R mutant cells. These results suggest that the RAD3 gene suppresses recombination involving short homologous sequences by promoting the degradation of the ends of broken DNA molecules.All organisms must repair the damage to their DNA that results from environmental stress and normal metabolism. Genetic recombination is one of several pathways that have evolved to repair this damage (13). This mode of repair can lead to deleterious consequences, however, because recombination between dispersed duplicate (ectopic) sequences can result in genome rearrangements or gene inactivation or both (42). These events are also implicated in human disease (20,24). Both DNA sequence length (2,26,51,53,58) and identity (6,18,36,43,45,47) are important determinants of the rate of recombination between repeated DNA sequences. What remains largely unclear is how short sequence length and mismatching hinder recombination. We took a genetic approach to studying the control of ectopic recombination in Saccharomyces cerevisiae. In a search for mutants that stimulate this recombination, we isolated an allele of the RAD3 gene (rad3-G595R) that increased the rate of recombination between sequences that share short lengths of perfect or imperfect homology.The RAD3 gene encodes a helicase (61) that is an essential component of the transcription and DNA repair complex TFIIH (factor B [10,66]) and is highly homologous to the human nucleotide excision repair gene XPD (60, 67). Many rad3 mutants have been isolated (13). These mutants exhibit a wide array of overlapping phenotypes including altered transcription (16), DNA repair (40,48,72), and recombination (37). One set of mutants, originally designated rem, were identified on the basis of their mutator and hyperrecombination phenotypes (14). The rem genes were subsequently found to be alleles of RAD3 and are thought to lead to the creation of recombinogenic double-strand breaks (37). Another mutant allele, rad3-2, is not hyperrecombinant but does decrease gene conversion tract length during intrachromosomal recombination (1). These diverse recombination phenotypes may be due to defects in the transcription of important recombination genes or to direct effects of mutant Rad3 proteins on recombination.White and Haber (71) analyzed the repair of double-strand breaks in DNA by homologous recombination between unlinked, duplicate sequences at the molecular level and showed that double-strand breaks are subject to extensive 5Ј-end degradation in wild-type cells. This processing is thought to be crucial for producing a single-stranded 3Ј end (3, 7...

Novel Function of Rad27 (FEN-1) in Restricting Short-Sequence Recombination

“…Similar to recombination in the rad3, ssl1, and ssl2 mutants described previously (2,3,29,34), SSR was stimulated severalfold in the rad27-null mutant, while recombination between long sequences was unaffected.…”

“…Since the mutant alleles partially complement the rad27 SSR phenotype (Table 5), we conclude that the endonuclease activities of Rad27 and FEN-1 are important for SSR control in yeast and, perhaps, human cells. While the effect of mutations in genes that encode TFIIH-NER core proteins on SSR correlates with defects in DSB processing (2,3,29,34), the loss of Rad27 has no impact on the exonucleolytic degradation of DSBs (data not shown). This suggests that Rad27 may restrict SSR by a mechanism that is different from that of the TFIIH-NER apparatus.…”

“…Previous work with several mutants demonstrated a correlation between increased DSB stability and enhanced frequencies of SSR (2,3,29,34). This led to the investigation of how null alleles of several nuclease genes affect SSR.…”

“…For example, following heteroduplex formation, Rad27 might remove unannealed 5Ј single strands generated by an advancing lagging-strand polymerase or helicase unwinding (33). The TFIIH-NER helicases Rad3 and Ssl2, which play a role in SSR control (2,3,29,34), and Dna2, a helicase that works in concert with Rad27 during DNA replication (9), are potential candidates for this activity. If unwinding is extensive, Rad27 could remove enough DNA to terminate recombination as discussed below.…”

“…Recent discoveries suggest a genetic basis for the limitation of short-sequence recombination (SSR) in yeast. Novel mutations in the RAD3, SSL1, and SSL2 genes, which encode components of the nucleotide excision repair (NER) apparatus and transcription factor TFIIH (14,69), disrupt the control of SSR and increase genome rearrangement (2,3,29,34). Genome instability may be due to slow removal of sequences at the ends of broken DNA molecules by an unidentified nuclease or nucleases.…”