Extensive DNA End Processing by Exo1 and Sgs1 Inhibits Break-Induced Replication

Abstract

Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination. DNA double-strand breaks (DSBs) can occur spontaneously in cells by defective DNA replication or are induced by various types of DNA damaging agents, such as those used in chemo- or radiation therapy. Failure to repair DSBs, or inappropriate repair, can result in chromosome loss or chromosome rearrangements, events associated with development of cancer cells. Typically, DNA DSBs have two ends that are reunited faithfully by copying from a homologous donor chromosome. For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome, a process called break-induced replication (BIR). This repair pathway is thought to be suppressed at two-ended DSBs to prevent extensive loss of heterozygosity. Here, we describe a new assay to physically monitor BIR to see how this repair pathway differs from the repair of two-ended DSBs. We show this pathway is infrequent, but can be detected by eliminating factors (Exo1 and Sgs1) that degrade linear DNA. However, increased chromosome rearrangements were found in the resection-defective strain. We found the kinetics of strand invasion in two-ended and one-ended DSB repair were the same, suggesting that the distinction between these pathways occurs after initial repair steps have begun.