Annealing of Complementary DNA Sequences During Double-Strand Break Repair inDrosophilaIs Mediated by the Ortholog of SMARCAL1

Abstract

DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesisdependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. We hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. We used unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesisdependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP-binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP-binding defect mutant showed reduced end joining, shedding light on the interaction between SDSA and end-joining pathways.

53BP1 nuclear body-marked replication stress in a human mammary cell model of BRCA2 deficiency

“…4C). Given our previous findings that HR is critical to suppress replication stress [14], the observed induction in 53BP1 nuclear bodies is reminiscent of a role of SMARCAL1 in HR, as has been reported in the fly system [49]. To determine the HR function of SMARCAL1 in MCF10A cells, we employed the DR-GFP reporter that is stably integrated as a single copy to assay HR activity [50,51] (Fig.…”

Mechanisms preventing Break-Induced Replication during repair of two-ended DNA double-strand breaks

“…Rad52 is highly conserved but in many eukaryotes it plays less prominent role in HR. In these organisms additional annealing enzymes evolved with a role in HR such as FANCA in human, annealing helicase SMARCAL1 in flies, or the BRCA2 homolog in U. maydis, Bhr2 (Benitez, Liu et al, 2018, Holsclaw & Sekelsky, 2017, Mazloum & Holloman, 2009). With the exception of Rad59, all the proteins studied here are well conserved between yeast and humans.…”

Loss of Drosophila Mei-41/ATR Alters Meiotic Crossover Patterning

“…In mitotic DSB repair, mei-41 mutants have no observable defects in the early steps of homologous repair of DSBs (e.g., resection, strand invasion, and repair synthesis), but Mei-41 appears to be required for the annealing or ligation steps of synthesis-dependent strand annealing (LaRocque et al 2007). Korda Holsclaw and Sekelsky (2017) hypothesized that Mei-41 activates Marcal1, which then catalyzes annealing of complementary sequences. Synthesis-dependent strand annealing promotes formation of noncrossover products, in contrast to the apparent role for Mei-41 in promoting crossovers during meiotic recombination.…”