RAD51-Dependent Break-Induced Replication Differs in Kinetics and Checkpoint Responses from RAD51-Mediated Gene Conversion

Abstract

Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time. However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms. Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome. Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G₂/M DNA damage checkpoint. RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process. RAD51-dependent BIR occurs efficiently in G₂-arrested cells. Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.

ACKNOWLEDGMENTS

We thank Moreshwar B. Vaze and present members of the Haber lab for their suggestions throughout this work and about the manuscript. This work was supported by National Institutes of Health grants GM20056 and GM61766 as well as National Science Foundation grant MCB0077257.