Distinct roles of RAD52 and POLQ in chromosomal break repair and replication stress response

Abstract

Disrupting either the DNA annealing factor RAD52 or the A-family DNA polymerase POLQ can cause synthetic lethality with defects in BRCA1 and BRCA2 , which are tumor suppressors important for homology-directed repair of DNA double-strand breaks (DSBs), and protection of stalled replication forks. A likely mechanism of this synthetic lethality is that RAD52 and/or POLQ are important for backup pathways for DSB repair and/or replication stress responses. The features of DSB repair events that require RAD52 vs. POLQ, and whether combined disruption of these factors causes distinct effects on genome maintenance, have been unclear. Using human U2OS cells, we generated a cell line with POLQ mutations upstream of the polymerase domain, a RAD52 knockout cell line, and a line with combined disruption of both genes. We also examined RAD52 and POLQ using RNA-interference. We find that combined disruption of RAD52 and POLQ causes at least additive hypersensitivity to cisplatin, and a synthetic reduction in replication fork restart velocity. We also examined the influence of RAD52 and POLQ on several DSB repair events. We find that RAD52 is particularly important for repair using ≥ 50 nt repeat sequences that flank the DSB, and that also involve removal of non-homologous sequences flanking the repeats. In contrast, POLQ is important for repair events using 6 nt (but not ≥ 18 nt) of flanking repeats that are at the edge of the break, as well as oligonucleotide microhomology-templated (i.e., 12–20 nt) repair events requiring nascent DNA synthesis. Finally, these factors show key distinctions with BRCA2, regarding effects on DSB repair events and response to stalled replication forks. These findings indicate that RAD52 and POLQ have distinct roles in genome maintenance, including for specific features of DSB repair events, such that combined disruption of these factors may be effective for genotoxin sensitization and/or synthetic lethal strategies.

Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling

“…Acute ATR inhibition was previously reported to impair RAD51 localization to IR-induced foci, which requires ATR-mediated phosphorylation of PALB2 and its subsequent interaction with BRCA1 ( 16 ). Since resection is only mildly affected upon acute ATR inhibition (Figure 2D ), we predicted that cells treated acutely with ATRi should still be proficient in utilizing other homology-directed repair mechanisms, such as repeat-mediated repair, which is highly dependent on RAD52 ( 51 ), but independent of the RAD51–PALB2–BRCA2 machinery ( 52 ). Consistent with this prediction, we found that acute VE-821 treatment impaired PARPi-induced RAD51 foci in U-2OS cells (Figure 3A and B ), but did not alter PARPi-induced RAD52 foci, likely reflecting alternative forms of DNA repair (Figure 3C and D ).…”

Ancestral Eukaryotes Reproduced Asexually, Facilitated by Polyploidy: A Hypothesis

“…[63] DSB repair pathway through SSA of long stretches of homologous sequences flanking the DSB site. [63] DRSR [64] RAD54 Works with RAD51 in HR [65] Required for mitotic diploid-specific recombination and repair in yeast [66] Enhances accessibility of DNA to other proteins and HR. [67] HR in DRSR [25] RPA Prevents premature association of RAD54 and HED1 with ssDNA [68] Prevents premature association of RAD54 and HED1 with ssDNA [68] ssDNA binding activity replaced by RAD51 in DNA repair [48] PMS1 Mismatch repair [69] Mismatch repair [46] -The reported functions of the genes in meiosis, in mitosis and their other functions are tabulated.…”

Physiological and Pathological Roles of RAD52 at DNA Replication Forks

“…Like NHEJ, SSA can also lead to joining the ends that belong to different chromosomes. Another process which has been recently demonstrated to involve RAD52 is MMEJ, which is another specialized DNA repair pathway that can act on resected DNA ends [39]. Similarly to the role in SSA, RAD52 has been shown to promote annealing of regions with >50 nt of homology especially in genomic regions flanked by extensive non-homologous sequences [39].…”

“…Another process which has been recently demonstrated to involve RAD52 is MMEJ, which is another specialized DNA repair pathway that can act on resected DNA ends [39]. Similarly to the role in SSA, RAD52 has been shown to promote annealing of regions with >50 nt of homology especially in genomic regions flanked by extensive non-homologous sequences [39]. As for SSA, also the function of MMEJ could be relevant to back-up the severe HR defect shown by BRCA1/2 deficient cells [39].…”

“…Similarly to the role in SSA, RAD52 has been shown to promote annealing of regions with >50 nt of homology especially in genomic regions flanked by extensive non-homologous sequences [39]. As for SSA, also the function of MMEJ could be relevant to back-up the severe HR defect shown by BRCA1/2 deficient cells [39].…”