Abstract
Stabilisation of stalled replication forks prevents excessive fork reversal and their pathological degradation, which can undermine genome integrity. Here we investigate a physiological role of RAD52 at stalled replication forks by using human cell models depleted of RAD52, a specific small-molecule inhibitor of the RAD52-ssDNA interaction, in vitro and single-molecule analyses. We demonstrate that RAD52 prevents excessive degradation of reversed replication forks by MRE11. Mechanistically, RAD52 binds to the stalled replication fork, promotes its occlusion and counteracts loading of SMARCAL1 in vitro and in vivo. Loss of the RAD52 function results in a slightly-defective replication restart, persistence of under-replicated regions and chromosome instability. Moreover, the RAD52-inhibited cells rely on RAD51 for completion of replication and viability upon replication arrest. Collectively, our data suggest an unexpected gatekeeper mechanism by which RAD52 limits excessive remodelling of stalled replication forks, thus indirectly assisting RAD51 and BRCA2 in protecting forks from unscheduled degradation and preventing genome instability.
Highlights
Recent findings explained how RAD51, BRCA1/2 and other factors contribute to reversed fork (RF) stabilisation, it is unknown how stalled forks are channelled to Replication fork reversal (RFR), since it was demonstrated that excessive RFR is as detrimental as its absence[3,4,5,15,16]
To study how RAD52 contributes to replication fork stability, we generated MRC5SV40 cells stably depleted of RAD52 by RNA interference (RNAi)
We performed nascent single-stranded DNA (ssDNA) detection by native iododeoxyuridine (IdU) immunofluorescence assay in shCTRL, shRAD52 cells or in the parental MRC5SV40 cells treated with the small-molecule RAD52 inhibitor epigallocatechin (EGC, RAD52i), which recapitulates the phenotype of shRAD52 cells[27]
Summary
Recent findings explained how RAD51, BRCA1/2 and other factors contribute to RF stabilisation, it is unknown how stalled forks are channelled to RFR, since it was demonstrated that excessive RFR is as detrimental as its absence[3,4,5,15,16]. RAD52 may be involved in the rescue of perturbed replication forks under pathological conditions Whether it contributes to the integrity of stalled forks in normal cells remained unknown. We find that RAD52 prevents super-physiological recruitment of fork reversal enzymes, including SMARCAL1, after replication fork arrest in vivo and antagonises SMARCAL1-mediated fork reversal in vitro. This new function of RAD52 is essential for a correct recovery from the replication arrest as RAD52 inhibition results in under-replication and a genuine reliance on RAD51 for viability. Our findings unveil a previously unidentified function of RAD52 in fork protection and recovery, which may be critical for genome integrity of normal cells and for the observed BRCA2–RAD52 synthetic lethal relationship
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