RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

Jim Westmoreland,Yan Yan,Anna Malkova,Kelly Van Hulle,Wenjian Ma,Michael A Resnick,Gregory P Copenhaver

doi:10.1371/journal.pgen.1000656

Abstract

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.

Highlights

Double-strand breaks (DSBs) in chromosomal DNA are common sources of genetic change that may be deleterious or beneficial to an organism
While a great deal is known about the mechanisms of both types of repair using model systems employing single, endonucleasegenerated DSBs [1,4], relatively little is known about the processes associated with repair of random spontaneous DSBs, as might occur during replication fork collapse, or damage-induced DSBs
To investigate molecular events surrounding the processing and repair of DSBs, we examined DSB induction and repair in haploid cells that were arrested by nocadazole in the G2/M phase of the cell cycle

Summary

Introduction

Double-strand breaks (DSBs) in chromosomal DNA are common sources of genetic change that may be deleterious or beneficial to an organism. They can arise as direct and processed lesions, errors during replication such as fork collapse, unprotected telomere ends, and they can be integral to programmed developmental processes including meiosis and immunoglobulin rearrangements. While a great deal is known about the mechanisms of both types of repair using model systems employing single, endonucleasegenerated DSBs [1,4], relatively little is known about the processes associated with repair of random spontaneous DSBs, as might occur during replication fork collapse, or damage-induced DSBs

Methods

Results

Conclusion