A nuclease-driven mechanism of post-replicative ssDNA gap suppression

During replication stress, failure to resolve post-replicative ssDNA gaps generated by PRIMPOL-mediated replication repriming is linked with chemosensitivity, and in all models reported to date the nuclease MRE11 has been implicated as a gap-promoting factor. We have dissected ssDNA gap dynamics following nucleoside analogue-mediated nascent strand termination and report a novel mechanism via which loss of the MRE11 negative regulator MRN-Interacting Protein (MRNIP) leads to MRE11 exonuclease-dependent suppression of post-replicative ssDNA gaps. This process is driven by UBC13 and the B-family TLS polymerase REV3L, suggesting that in the absence of MRNIP, dysregulated MRE11 activity at chain termination sites licenses gap filling via template switching. In the absence of PRIMPOL-dependent repriming, MRNIP loss leads to paradoxical SMARCAL1, MRE11 and primosome-dependent ssDNA gaps, suggesting that nucleolytic digestion of reversed forks generates DNA intermediates that platform primase redundancy in the context of chain termination. We also highlight novel site-specific roles for the anti-resection factor 53BP1 in enabling primase redundancy via suppression of the nuclease EXO1, and in limiting the EXO1-dependent processing of post-replicative ssDNA gaps. Finally, we demonstrate that CDK-dependent MRNIP phosphorylation is required for MRNIP functionality in the regulation of ssDNA gaps and sensitivity to chain terminators. This work represents the first report of nuclease-driven post-replicative gap filling, illuminates an additional level of versatility in the replication stress response, and expands our understanding of the context-dependent links between nuclease regulation and chemoresistance.