The Effect of Repeat Length on Marcal1-Dependent Single-Strand Annealing in Drosophila

Abstract

Proper repair of DNA double strand breaks (DSBs) is essential to maintenance of genomic stability and avoidance of genetic disease. Organisms have many ways of repairing DSBs, including use of homologous sequences through homology-directed repair (HDR). While HDR repair is often error-free, in single-strand annealing (SSA) homologous repeats flanking a DSB are annealed to one another, leading to deletion of one repeat and the intervening sequences. Studies in yeast have shown a relationship between the length of the repeat and SSA efficacy. We sought to determine the effects of homology length on SSA in Drosophila, as Drosophila uses a different annealing enzyme (Marcal1) than yeast. Using an in vivo SSA assay, we show that 50 base pairs (bp) is insufficient to promote SSA and that 500-2000 bp is required for maximum efficiency. Loss of Marcal1 generally followed the same homology length trend as wild-type flies, with SSA frequencies reduced to about a third of wild-type frequencies regardless of homology length. Interestingly, we find a difference in SSA rates between 500 bp homologies that align to the annealing target either nearer or further from the DSB, a phenomenon that may be explained by Marcal1 dynamics. This study gives insights into Marcal1 function and provides important information to guide design of genome engineering strategies that use SSA to integrate linear DNA constructs into a chromosomal DSB.