Multiple-Pathway Analysis of Double-Strand Break Repair Mutations in Drosophila

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Abstract
The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage “decision circuit” in which the DSB is first placed into one of two pools from which a specific pathway is then selected. DNA is a fragile thread that often breaks. When it does, the cell must find a way to splice the broken ends back together in order to continue its cycle of replication. Cells possess an array of ways to rejoin broken DNA ends, each with advantages and disadvantages. Some are “quick and dirty,” sacrificing accuracy for robustness. They do the basic job of resealing the break but often result in random base changes at the site of the repair. At the other extreme are methods with greater fidelity but added restrictions, such as requiring chromosome replication. We used an experimental system to obtain highly accurate measurements of the relative usage of various repair methods in developing germ cells of fruit flies. The measurements were made in normal flies as well as those carrying mutations at each of 11 genes involved in DNA repair. Most previous studies of these genes focused on specific biochemical pathways. Our results looked at how the repair apparatus as a whole compensates for defects in individual components. The data point to a “decision circuit” for matching each break to a repair method and provide new insight into how our DNA repair machinery protects the genome's integrity.