Spatiotemporal dynamics of homologous recombination repair at single collapsed replication forks

Abstract

Homologous recombination (HR) is a crucial pathway for the repair of DNA double-strand breaks. BRCA1/2 breast cancer proteins are key players in HR via their mediation of RAD51 nucleofilament formation and function; however, their individual roles and crosstalk in vivo are unknown. Here we use super-resolution (SR) imaging to map the spatiotemporal kinetics of HR proteins, revealing the interdependent relationships that govern the dynamic interplay and progression of repair events. We show that initial single-stranded DNA/RAD51 nucleofilament formation is mediated by RAD52 or, in the absence of RAD52, by BRCA2. In contrast, only BRCA2 can orchestrate later RAD51 recombinase activity during homology search and resolution. Furthermore, we establish that upstream BRCA1 activity is critical for BRCA2 function. Our analyses reveal the underlying epistatic landscape of RAD51 functional dependence on RAD52, BRCA1, and BRCA2 during HR and explain the phenotypic similarity of diseases associated with mutations in these proteins.

XAB2 prevents abortive recombinational repair of replication-associated DNA double-strand breaks and its loss is synthetic lethal with RAD52 inhibition

“…RAD52 acts as mediator of ssDNA/RAD51 filaments during seDSB repair by HR in human cells, a role where it can be replaced by BRCA2 [14]. The severe TMZ sensitivity induced by loss of RAD52 and its strong ability to rescue the defective phenotypes associated with XAB2 depletion suggest that RAD52 also exerts a function independently of RAD51, in line with a previous report [15].…”

“…8). As XAB2 is present at seDSBs, we propose that XAB2 acts 14 via a direct mechanism to promote Ku eviction from resected seDSBs. The molecular details of this novel pathway and its interplay with the ATM-dependent pathway in counteracting Ku accumulation merit further exploration.…”

“…These filaments mediate homology search and strand invasion into the homologous sister chromatid, generating a displacement loop (Dloop) [13]. The single-strand annealing (SSA) factor RAD52 facilitates the assembly of ssDNA/RAD51 nucleoprotein filaments during HR-mediated seDSB repair in human cells [14]. Similar to yeast RAD52 [13], human RAD52 can also promote BIR mechanisms 5 without RAD51.…”

Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

“…Validating U-DNA-Seq data, we found that U-DNA staining shows significant colocalization with staining for both chromatin markers; H3K36me3 and H3K27me3, based on cross-pair correlation analysis of the super-resolution images as shown in Figure 7C-D. The rate of colocalization, as determined by the interaction factor (IF) value (Bermudez-Hernandez et al, 2017; Whelan et al, 2018), was statistically significant between the uracil signal and both chromatin markers in each case of drug treatment, when compared to the non-treated sample as well as to a generated set of random distribution patterns of these chromatin markers.…”

“…The interaction factor (IF) quantifies the colocalization of red and green foci within a cell nucleus by measuring the area of overlap between the two sets of foci (Bermudez-Hernandez et al, 2017; Whelan et al, 2018). The positions of the green foci are then randomized and the overlap between the two colours is measured again.…”