Mre11 complex links sister chromatids to promote repair of a collapsed replication fork

Abstract

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.

Keywords: Mre11; Rad50; double-strand break repair; genome maintenance; recombination.

Fig. 1.

Mre11–Rad50 complexes. Composition of Mre11–Rad50 complexes and their proposed associations with DSBs and a SC repair template are shown.

Fig. 2.

Rad50 ChIP assays with the seDSB and unbroken sister chromatid at a collapsed replication fork. (A) Diagram of the mat1 locus showing the site of fork collapse and locations of PCR products used for ChIP assays. (B) Rad52 ChIP enriches the mat1 seDSB site (BE) and SC region, while Ku ChIP only enriches the BE region. Assays were performed with Rad52-5FLAG or pKu70-3HA expressed from their endogenous loci in mat2,3∆ backgrounds. The smt0⁻ control strain has a 263-bp deletion at the mat1 locus that prevents SSB formation. (C) Rad50 ChIP enriches the BE and SC sites. ChIP assays were performed with TAP-Rad50 expressed from the endogenous locus. The swi3∆ strain is defective for formation of the SSB. (D) Rad52 ChIP enrichments at the BE and SC sites are decreased in swi3∆ cells. ChIP assays were performed with Rad52-5FLAG. (E) Rad50 ChIP enrichments at the BE and SC sites occur in S-phase. ChIP assays were performed with TAP-Rad50 in a mat2,3∆ cdc25-22 background. Cells were arrested in G2-phase by incubating at 35.5 °C for 4 h and were then shifted to 25 °C to resume cell cycle progression. The septation index correlates with entry into S-phase. Bars indicate mean ± SEM of triplicate samples. Similar results were observed in three (B and E) or two (C and D) independent experiments.

Fig. 3.

Rad50 BE and SC ChIP interactions occur sequentially. ChIP assays were performed with TAP-Rad50 and Rad52-5FLAG in a mat2,3∆ cdc25-22 background. Samples were taken between 70 and 90 min after release from the cdc25-22 G2 arrest. Bars indicate mean ± SEM of triplicate samples. Similar results were observed in three independent experiments.

Fig. 4.

Provision of an intrachromosomal repair template partially suppresses the mat1 seDSB repair defect in rad50∆ pku80∆ cells. Colonies of the indicated genotypes were obtained by tetrad dissection of rad50∆ pku80∆ h⁻ crossed to h⁹⁰ or mat2,3∆ cells. Colony size (growth) was measured and normalized to WT h⁻ as described in Materials and Methods. (A) Unimpaired growth of h⁹⁰ and mat2,3∆ strains in the WT (rad50⁺ pku80⁺) background. (B) Equal growth of h⁹⁰ and mat2,3∆ strains in the pku80∆ background indicates that intrachromosomal SDSA (h⁹⁰) repair and interchromosomal SCR (mat2,3∆) repair of the mat1 collapsed fork are equally efficient in the absence of Ku. (C) Improved growth of h⁹⁰ versus mat2,3∆ strains in the rad50∆ pku80∆ background indicates that the presence of the intrachromosomal repair template in h⁹⁰ cells partially alleviates the requirement for Rad50 to repair the mat1 seDSB. Bars are mean ± SEM (n ≥ 3).

Fig. 5.

Analysis of Rad50 hook mutants. (A) Sequence alignment of the central portion of the Rad50 hook domain showing the conserved CXXC motif. The mutants are highlighted in green. (B) The rad50-C1G and rad50-C2G mutants retain partial resistance to DNA damaging agents. Fivefold serial dilutions of cells were plated on the indicated concentrations of camptothecin (CPT) or hydroxyurea (HU), or exposed to the indicated doses of IR before plating. (C) Reductions of Rad50 abundance in the rad50-C1G and rad50-C2G mutants. IP, immunoprecipitation; TUBLIN, tubulin. (Top) Immunoblot (IB) of immunoprecipitated TAP-Rad50. 5×, fivefold greater sample loading in mutants relative to WT. (Bottom) Antitubulin IB of whole-cell extracts (WCE). (D) Yeast two-hybrid interactions of WT and mutant Rad50 with Mre11. The leftmost panel (-Leu -Trp) selects for the reporter plasmids. The other panels indicate yeast two-hybrid interactions, with increasing stringency occurring right to left. (E) Rad50 hook mutants ChIP to a HO-induced DSB. Experiments were performed in ctp1∆ backgrounds to eliminate resection (6). Derepression of the thiamine (B1)-repressible nmt1 promoter that controls HO expression requires about 18–20 h of growth in −B1 media (media in which expression of HO endonuclease is induced). Samples were taken at 22 and 24 h. The ratio is shown for Rad50 enrichment normalized to cut efficiency as determined by PCR amplification across the HO cut site (6). The ratio in the presence of B1 was set to 1. Bars indicate mean ± SEM of triplicate samples. Similar results were observed in two independent experiments. (F) Zinc coordination by Rad50 is essential for Tel1 activity. Immunoblot of γ-H2A and total histone H2A with or without treatment with 90 Gy of IR. Assays were performed in rad3∆ backgrounds because both Rad3/ATR and Tel1/ATM phosphorylate histone H2A at DSBs (41).

Fig. 6.

Rad50 zinc hook is critical for SCR. (A) Tetrad analysis reveals synthetic lethality of rad50-C1G and rad50-C2G with mutants lacking Rad2, which is the FEN-1 nuclease ortholog. (B) Tetrad analysis reveals synthetic lethality of rad50-C1G and rad50-C2G with cdc17-K42. The DNA ligase I ortholog is cdc17. Spores were germinated at 25 °C, which is the permissive temperature for cdc17-K42. (C) The rad50-C1G and rad50-C2G mutations are synthetic lethal with mat2,3∆. This lethality is suppressed by pku80∆. (D) Improved growth of h⁹⁰ versus mat2,3∆ strains in the rad50-C1G pku80∆ and rad50-C2G pku80∆ backgrounds indicates that the presence of the intrachromosomal repair template in h⁹⁰ cells partially alleviates the requirement for the Rad50 zinc hook to repair the mat1 collapsed fork. Colony size (growth) was measured and normalized to WT h⁻ as described in Materials and Methods. Bars are mean ± SEM (n ≥ 3).