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.

Transgene removal using an in cis programmed homing endonuclease via single-strand annealing in the mosquito Aedes aegypti

“…The threshold requirements for efficient SSA are likely to vary based on host organism, chromosomal loci, lengths and arrangements of direct repeats, and resection distances [55][56][57][58][59][60] . Intrachromosomal deletions mediated by the SSA pathway can result in deletions of at least 80-kb at high efficiency 34 with the length of repeats and distance of separation strongly influencing this mode of repair in Drosophila melanogaster 61 , Saccharomyces cerevisiae 34 , and vertebrate cells 35 .…”

“…In Drosophila , in vivo DSB reporter assays showed that CtIP was critical for efficient SSA under short (~550 bp) and extensive (~3.6 kb) distance of resection 52 , suggesting that the initial generation of ssDNA may be a rate-determining process. Similarly, loss of Marcal1, an annealing factor that binds to ssDNA–dsDNA interfaces 53 , 54 , reduced SSA frequencies compared to wild type regardless of homology length 55 .…”

“…Moreover, it was suggested that a competition can occur between two pairs of DRs, and a preference can be made based on their relative sizes and proximity to the DSB 57 . Another assay revealed that SSA rates were enhanced when 500-bp homologies were aligned to be the annealing target farther to the DSB site than closer 55 . In this work, we hypothesized that a larger DR motif may be more favorable for SSA-based transgene elimination.…”