Edited by Richard D. Kolodner, Ludwig Institute for Cancer Research, La Jolla, CA, and approved July 12, 2018 (received for review May 15, 2018)
When replication forks collapse at single-strand DNA breaks, the resulting single-ended DNA double-strand breaks (seDSBs) are repaired by sister chromatid recombination (SCR). The Mre11–Rad50–Nbs1 (MRN) protein complex and CtIP/Ctp1 nucleolytically resect the seDSB to initiate SCR, but MRN was also proposed to promote SCR by tethering the seDSB to the unbroken sister chromatid. Here, we show that MRN sequentially localizes with the seDSB and sister chromatid during SCR. Provision of an intrachromosomal repair template alleviates the requirement for the nonnucleolytic function of MRN in repair of a collapsed replication fork. Zinc-coordinating “hooks” in Rad50 that are linchpins for MRN intercomplexes are essential for SCR. These studies support the MRN tethering hypothesis.
Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11–Rad50–Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre112–Rad502 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid.
↵1M.Z. and H.Z. contributed equally to this work.
Author contributions: M.Z., H.Z., and P.R. designed research; M.Z., H.Z., and O.L. performed research; M.Z. and H.Z. contributed new reagents/analytic tools; M.Z., H.Z., O.L., and P.R. analyzed data; and M.Z., H.Z., and P.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
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