RAD52 Prevents Excessive Replication Fork Reversal and Protects from Nascent Strand Degradation

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 singlemolecule 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 underreplicated 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.

Rad52 Restrains Resection at DNA Double-Strand Break Ends in Yeast

“…It will be of interest to determine whether Rad52 in vertebrate organisms similarly protects DNA ends from extensive degradation. Recent study implicates that indeed human RAD52 could protect stalled replication forks from degradation (Malacaria et al, 2019).…”

A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability

“…Mre11 Suppresses Oncogene-Induced R-Loops and DNA Damage The Mre11 complex suppresses the accumulation of spontaneous, replication-associated DSBs (Oh and Symington, 2018). In contrast, some studies have demonstrated that Mre11 nuclease activity can also promote destabilization of stalled replication forks (Malacaria et al, 2019;Schlacher et al, 2011). To determine whether Mre11 promotes or suppresses oncogene-induced DSBs in pMMECs, we measured gH2AX foci, 53BP1 foci, and neutral COMET tails (Figures 4A, 4B, and S4A).…”

Sequential role of RAD51 paralog complexes in replication fork remodeling and restart

“…Importantly, this deregulated degradation of nascent DNA is also genetically dependent on fork remodeling, as regressed arms are mandatory entry points for DNA degrading enzymes 13 , 14 , 22 , 23 . Moreover, the HR protein RAD52 was recently shown to modulate fork stability and restart, by limiting SMARCAL1-dependent fork reversal or regulating reversed fork processing by MRE11 14 , 24 . Collectively, these recent observations have implicated the RAD51 recombinase and other HR factors both in the formation and in the stability of reversed replication forks 5 , 14 .…”