A novel assay for break-induced replication reveals a role for resection.

摘要

Homology-dependent repair of DNA double-strand breaks usually occurs by a conservative gene conversion mechanism. However, some DSBs present only one end and are repaired by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive loss of heterozygosity, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequences 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 homologous sequences on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR is inefficient in wild type cells due to rapid degradation of the linearized vector. However, in exo1Delta, sgs1Delta and exo1Delta sgs1Delta mutants, which are defective in the 5'-3' resection of DSBs, the frequency of BIR is increased by 3 to 40-fold. Extension of the invading end of the chromosome fragment vector (CFV) is detectable two hours after induction of the I-SceI endonuclease in the exo1Delta, sgs1Delta and exo1Delta sgs1Delta mutants, but formation of fully repaired products is delayed by several hours. The rare chromosome rearrangements due to BIR template switching at repeated sequences are increased in sgs1Delta mutants, possibly due to increased crossing over and/or pairing between diverged sequences. The resection-defective strains also resulted in more efficient gene conversion in a two-ended repair assay suggesting extensive degradation is inhibitory to both BIR and gene conversion from limited homology substrates. The kinetics of strand invasion were the same in the BIR and gene conversion assay suggesting the initial step of these processes are the same, and the decreased efficiency of BIR is due to a defect at a later step.