Evidence for Multiple Cycles of Strand Invasion During Repair of Double-Strand Gaps in Drosophila

Abstract

DNA double-strand breaks (DSBs), a major source of genome instability, are often repaired through homologous recombination pathways. Models for these pathways have been proposed, but the precise mechanisms and the rules governing their use remain unclear. In Drosophila, the synthesis-dependent strand annealing (SDSA) model can explain most DSB repair. To investigate SDSA, we induced DSBs by excision of a P element from the male X chromosome, which produces a 14-kb gap relative to the sister chromatid. In wild-type males, repair synthesis tracts are usually long, resulting in frequent restoration of the P element. However, repair synthesis is often incomplete, resulting in internally deleted P elements. We examined the effects of mutations in spn-A, which encodes the Drosophila Rad51 ortholog. As expected, there is little or no repair synthesis in homozygous spn-A mutants after P excision. However, heterozygosity for spn-A mutations also resulted in dramatic reductions in the lengths of repair synthesis tracts. These findings support a model in which repair DNA synthesis is not highly processive. We discuss a model wherein repair of a double-strand gap requires multiple cycles of strand invasion, synthesis, and dissociation of the nascent strand. After dissociation, the nascent strand may anneal to a complementary single strand, reinvade a template to be extended by additional synthesis, or undergo end joining. This model can explain aborted SDSA repair events and the prevalence of internally deleted transposable elements in genomes.

Mutator Phenotype ofCaenorhabditis elegansDNA Damage Checkpoint Mutants

“…Thus, the e2825 deletion may have been created by a break in the Helitron transposable element at the unc-58 locus, strand invasion of a homologous transposon on C11G10, and dissociation and end-joining at the unc-58 locus to seal the break. These results suggest that C. elegans checkpoint-defective mutants may be deficient for a model of HR-dependent break repair known as synthesis-dependent strand annealing, which may involve processive cycles of template-directed DNA synthesis followed by dissociation to search for homology with the other end of the break (Nassif et al 1994;McVey et al 2004a). Recombination events similar to that of the e2825 deletion have been observed for DNA sequences flanking P elements in Drosophila (Tsubota and Huong 1991;Heslip et al 1992).…”

Differential Usage of Alternative Pathways of Double-Strand Break Repair in Drosophila

“…All the experiments presented here examine events in the male germ line while some earlier experiments have looked at the female germ line ( Johnson-Schlitz and Engels 1993). Previous work has investigated other aspects of DSB repair that we have not tested with Rr3 such as the length of conversion tracts (Gloor et al 1991), the effects of polymorphisms (Nassif and Engels 1993;Dray and Gloor 1997;Coveny et al 2002), the repair efficiency of large gaps (Nassif et al 1994;McVey et al 2004a), and the preference hierarchy of template location (i.e., sister chromatid vs. allelic vs. ectopic sites in cis or in trans) (Johnson-Schlitz and Engels 1993; Engels et al 1994;Keeler et al 1996;Lankenau et al 1996;Lankenau and Gloor 1998).…”

RECQ1 Helicase Interacts with Human Mismatch Repair Factors That Regulate Genetic Recombination*[boxs]

“…4 It is conceivable that RECQ1 shares with BLM and perhaps other RecQ helicases a specialized function in a subpathway of homologous recombination such as synthesis-dependent strand annealing. Recently, a genetic role for BLM in synthesis-dependent strand annealing was evidenced (54,55). In the model presented, BLM is predicted to function downstream of strand invasion to unwind a D-loop intermediate to free the newly synthesized strand.…”