DNA strand-exchange patterns associated with double-strand break-induced and spontaneous mitotic crossovers in Saccharomyces cerevisiae.

Yee Fang Hum,Sue Jinks-Robertson

doi:10.1371/journal.pgen.1007302

Abstract

Mitotic recombination can result in loss of heterozygosity and chromosomal rearrangements that shape genome structure and initiate human disease. Engineered double-strand breaks (DSBs) are a potent initiator of recombination, but whether spontaneous events initiate with the breakage of one or both DNA strands remains unclear. In the current study, a crossover (CO)-specific assay was used to compare heteroduplex DNA (hetDNA) profiles, which reflect strand exchange intermediates, associated with DSB-induced versus spontaneous events in yeast. Most DSB-induced CO products had the two-sided hetDNA predicted by the canonical DSB repair model, with a switch in hetDNA position from one product to the other at the position of the break. Approximately 40% of COs, however, had hetDNA on only one side of the initiating break. This anomaly can be explained by a modified model in which there is frequent processing of an early invasion (D-loop) intermediate prior to extension of the invading end. Finally, hetDNA tracts exhibited complexities consistent with frequent expansion of the DSB into a gap, migration of strand-exchange junctions, and template switching during gap-filling reactions. hetDNA patterns in spontaneous COs isolated in either a wild-type background or in a background with elevated levels of reactive oxygen species (tsa1Δ mutant) were similar to those associated with the DSB-induced events, suggesting that DSBs are the major instigator of spontaneous mitotic recombination in yeast.

Highlights

DNA is constantly assaulted by endogenous damaging agents and maintenance of its integrity is essential for the stable inheritance of genetic material
Chromosome breakage during mitosis is a threat to genome stability, and duplex integrity can be restored through the process of homologous recombination
The goals of the current work were (1) to define heteroduplex DNA (hetDNA) patterns associated with DSBinduced CO events and (2) to use the Double-strand breaks (DSBs)-associated hetDNA patterns to infer whether spontaneous- and reactive oxygen species (ROS)-initiated CO events are most often initiated by DSBs or by single-strand nicks/gaps

Summary

Introduction

DNA is constantly assaulted by endogenous damaging agents and maintenance of its integrity is essential for the stable inheritance of genetic material. Double-strand breaks (DSBs), which disrupt both DNA strands, are especially toxic lesions whose repair can result in local sequence changes as well as structural alterations. DSBs can be repaired by homologous recombination (HR), which utilizes an intact duplex as a repair template and is considered a high-fidelity repair mechanism. DSBs can be repaired by the nonhomologous end-joining (NHEJ) pathway, which processes and directly ligates the broken ends and is error prone relative to HR (reviewed by [1]). Single-strand breaks (SSBs), such as nicks and gaps, affect only one strand of DNA and are orders of magnitude more abundant than DSBs [2]. SSBs are natural intermediates during lagging-strand replication, during excision-repair reactions, and during topoisomerase-mediated relief of DNA torsional stress

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