ERCC1 and MUS81–EME1 promote sister chromatid separation by processing late replication intermediates at common fragile sites during mitosis

Abstract

Chromosomal instability (CIN) is a hallmark of tumour initiation and progression. Some genomic regions are particularly unstable under replication stress, notably common fragile sites (CFSs) whose rearrangements in tumour cells contribute to cancer development. Recent work has shown that the Fanconi anaemia (FANC) pathway plays a role in preventing defective chromosome segregation and CIN under conditions of replication stress. Strikingly, FANCD2 is recruited to regions hosting CFSs on metaphase chromosomes. To decipher the mechanisms protecting CFSs in G2/M, we searched for proteins that co-localize with FANCD2 on mitotic chromosomes, and identified XPF-ERCC1 and MUS81-EME1, two structure-specific endonucleases. We show that depletion of either ERCC1 or MUS81-EME1 affects accurate processing of replication intermediates or under-replicated DNA that persist at CFSs until mitosis. Depletion of these endonucleases also leads to an increase in the frequency of chromosome bridges during anaphase that, in turn, favours accumulation of DNA damage in the following G1 phase.

A Mechanism for Controlled Breakage of Under-replicated Chromosomes during Mitosis

“…We show that EME2 depletion and PLK1 inhibition efficiently restore the bulk of DNA replication in cells treated with WEE1 inhibitors, arguing that a significant fraction of replication intermediates targeted for cleavage is capable of supporting DNA synthesis effectively. Therefore, even though most naturally-occurring MUS81 substrates are likely to be stalled RFs (Beck et al, 2012;Naim et al, 2013;Neelsen et al, 2013;Ying et al, 2013), we envision that RF remodeling may not be a prerequisite for substrate recognition and cleavage. In agreement with this model, WEE1 inhibition triggers massive DNA breakage and chromosome pulverization without significantly altering the recruitment of RPA to replicating chromosomes ( Figure 3H and I).…”

“…MUS81 is known to associate with chromatin at under-replicated regions during mitosis (Naim et al, 2013;Ying et al, 2013). Since MUS81-SLX4 association occurred with similar timing, we examined if SLX4 also binds chromatin in mitosis and whether it plays a role in MUS81 recruitment.…”

“…Previous studies revealed a key role for MUS81 endonuclease in the resolution of stalled replication intermediates at the onset of mitosis (Naim et al, 2013;Ying et al, 2013). However, whether and how cells prevent MUS81 from cleaving vital DNA structures during unperturbed S-phase was unclear.…”

“…In agreement with this notion, MUS81 has been implicated in the nucleolytic processing of stalled RFs after prolonged S-phase arrest . Moreover, by cleaving persistent replication intermediates during M-phase, MUS81 promotes DNA repair synthesis outside S-phase and safeguards faithful chromosome segregation (Minocherhomji et al, 2015;Naim et al, 2013;Ying et al, 2013).…”

A new class of ultrafine anaphase bridges generated by homologous recombination

“…treatment with the DNA polymerase inhibitor aphidicolin). The Fanconi anemia proteins, FANCI and FANCD2 associate with CFSs after replication stress and localize to the termini of FS-UFBs [8,12–14]. Finally, telomeric UFBs (T-UFBs) can be induced by interfering with the replication of telomeres or by overexpression of the shelterin component TRF2 that induces chromosome end-to-end fusions [15–17].…”