Abstract
SLX4 is a versatile protein serving as docking for multiple structure-specific endonucleases during DNA repair, however, little is known about its function at demised replication forks. Using RNAi or FA-P cells complemented with SLX4 mutants that abrogate interaction with MUS81 or SLX1, we show that SLX4 cooperates with MUS81 to introduce DSBs after replication stress but also counteracts pathological targeting of demised forks by GEN1. Such unexpected function of SLX4 is unrelated to interaction with endonucleases, but concerns the physical presence of the protein. Strikingly, ectopic expression of the Holliday junction-binding protein RuvA inhibits DSBs in SLX4-deficient cells by preventing GEN1 chromatin-association, and rescues proliferation and genome integrity upon replication stress. Altogether, our results indicate that SLX4 is crucial to prevent accidental processing of Holliday junction-like intermediates at demised forks also suggesting that spontaneous genome instability in FA-P cells may derive, at least partially, from unscheduled action of GEN1 in S-phase.
Highlights
During mitotic processing of recombination intermediates, another SSE, GEN1(Yen1), can substitute for MUS81 or SLX416,17
We show that SLX4 is necessary to support MUS81 function at demised replication forks, even if this crucial role is masked by the take-over of GEN1-dependent DSBs
GEN1-dependent DSBs observed in absence of SLX4 maintain the same genetic dependency on RAD52 as that formed by MUS81
Summary
During mitotic processing of recombination intermediates, another SSE, GEN1(Yen1), can substitute for MUS81 or SLX416,17. Even though GEN1(Yen1) shows ability to target replication intermediates in vitro, its activity appears strictly prevented in S-phase in both yeast and human cells through CDK-dependent phosphorylation and nuclear exclusion[6]. We analysed the contribution of SLX4 and GEN1 to replication fork processing into DSBs under pathological replication stress induced by CHK1 inhibition or oncogene activation. Produce DSBs after pathological replication stress in absence of MUS81, contributing to cell survival, they indicate that such DSBs are prevented by the presence of SLX4, even if no other SSE is available to target demised replication forks. Our findings contribute to shed light into pathological processing of perturbed replication forks in human cells They may be useful to our understanding of the correlation between replication stress and genomic instability in cancer as well as in genetic diseases
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