The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks

Abstract

Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair.

FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress

“…However, the precise substrate recognized by MUS81 at the fork is not known, but possibilities include Holliday junctions generated by replication fork regression or the stalled fork itself 8,21,22 . Our data on the role of MUS81 in the response to HU are in agreement with Hanada et al 6 with regard to MUS81-induced DNA DSB formation. Yet, Hanada et al 6 found that genetic ablation of MUS81 led to HU-sensitivity.…”

“…We found that DNA DSB formation was evident after treatment for 24 h as evidenced by pulsed-field gel electrophoresis (PFGE) that detects DNA breaks (Fig. 1c), a finding that is in agreement with a number of previous studies 6,15 . Furthermore, treatment with HU led to checkpoint activation at the time of DNA DSB accumulation, including robust ATM activation and the phosphorylation of key downstream targets like CtIP and CHK2 (Fig.…”

“…The mechanisms by which stalled forks are processed and the biological role of this processing are not well understood in mammalian cells. Replication arrest does not induce detectable numbers of DNA DSBs at early times after treatment, but these breaks do arise at later stages induced by HU or aphidicolin (around 18-24 h after treatment) 15 , where the fork is subjected to cleavage by the MUS81 endonuclease 6 . Our data have uncovered a critical role for FBH1 in MUS81-induced cleavage of stalled replication forks, as DSBs were not detected in FBH1-depleted cells following HU treatment.…”

“…Prolonged HU treatment leads to the collapse of stalled replication forks accompanied by DNA breakage mediated by the MUS81 endonuclease 6 . Ablation of MUS81, therefore, prevents the accumulation of DSBs following HU treatment.…”

“…Following prolonged replication arrest, the disassembly of the replication machinery can be accompanied by the cleavage of the fork by the structure-specific endonuclease MUS81 (ref. 6). This generates a one-ended DSB that is thought to be the precursor for recombination-dependent replication fork restart 7,8 .…”