Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination

Abstract

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.

Figure 1

The Smc5–Smc6 complex is enriched around a HO-induced DSB during G2–M. (a) Binding of Smc6 around the MAT locus on chromosome III in JKM179 cells (Smc6–9Myc), without (uncut) or with (cut) a DSB at MAT, in cells arrested at different stages of the cell cycle. Cells were grown at 30 °C, arrested and galactose was added to half of the cells (cut), before being processed for ChIP. Input DNA and DNA coimmunoprecipitated with anti-Myc antibody (IP) were amplified using primer sets corresponding to sequences different distances from the MAT cut. (b) NHEJ assays with transient HO induction. Percentage cell survival is shown. WT, wild type. (c) MAT switching assays in wild-type and smc5–smc6 mutant strains. Percent switching was calculated from the ratio of the amount of HO cleavage at 1 h compared with the amount of final product, normalized to the MAT distal DNA in each lane. s.d. from 3 independent experiments are shown in b and c.

Figure 2

Inactivation of Smc5–Smc6 reduces DSB repair by SCR. (a) Quantification of HO-induced DSB repair by SCR in wild-type, nse5-1 and smc6-9 cells. DSB disappearance (left) and SCR accumulation (right) are shown. A schematic representation and Southern blot analysis of recombination intermediates are shown in the Supplementary Information, Fig. S2a, b. (b) Quantification of HO-induced DSB repair by SCE in wild-type, nse5-1 and smc6-9 cells. DSB disappearance (left) and accumulation of SCE products (right) are shown. A schematic representation and Southern blot analysis of recombination intermediates are shown in the Supplementary Information, Fig. S2c, d. The average of 3 experiments with s.d. are shown in a and b. (c) Increased GCR formation in smc6-9 mutants. The smc6-9 strain is isogenic with the wild-type strain, RDKY3615 [ura3-52, leu2Δ1, trp1Δ63, his3Δ200, lys2ΔBgl, hom3-10, ade2Δ1, ade8, YEL069::URA3]. The numbers in parenthesis indicate the fold induction of GCR rate relative to the wild-type GCR rate. Can^r, canavanine resistance; 5-FOA^r, 5-fluoracetic acid resistance.