The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection

Abstract

Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5'-3' degradation of DSB ends that yields the 3' single-stranded DNA required for the loading of checkpoint and recombination proteins. In yeast, the Mre11-Rad50-Xrs2 complex (Xrs2 is known as NBN or NBS1 in humans) and Sae2 (known as RBBP8 or CTIP in humans) initiate end resection, whereas long-range resection depends on the exonuclease Exo1, or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 together with the endonuclease Dna2 (refs 1-6). DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood. Here we show that the yeast Saccharomyces cerevisiae Fun30 protein and its human counterpart SMARCAD1 (ref. 8), two poorly characterized ATP-dependent chromatin remodellers of the Snf2 ATPase family, are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates the repair of camptothecin-induced DNA lesions, although it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs, and the kinetics of recruitment is similar to that of EXO1. The loss of SMARCAD1 impairs end resection and recombinational DNA repair, and renders cells hypersensitive to DNA damage resulting from camptothecin or poly(ADP-ribose) polymerase inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodellers in controlling end resection, homologous recombination and genome stability in the context of chromatin.

Figure 1. fun30Δ and DNA end-resection mutants show high BIR efficiencies

BIR efficiencies of selected homozygous diploid null mutants relative to wild type (WT; BY4743). Mutants have been ranked according to their BIR efficiencies. Two BIR experiments using transformations of mutant pools were performed (Supplementary Fig. 1). The rank of each mutant in these two BIR experiments is given in parentheses. This rank is bottom-up for mutants with BIR efficiencies lower than wild type, and top-down otherwise. A schematic of the BIR assay is provided in the box. Error bars denote ± mean absolute deviation of two independent experiments.

Figure 2. Fun30 promotes long-range 5′-3′ DNA end resection and is recruited to DSBs

a, Southern blot analysis of StyI (S)/BstXI (B)-digested genomic DNA after alkaline gel electrophoresis. r1 to r7 fragments are partially ssDNA fragments. b, As in a, except that exo1Δ mutants were MATalpha strains, showing a longer uncut fragment (1.9 kb). c, Southern blot analysis of StyI-digested genomic DNA after alkaline gel electrophoresis to monitor ssDNA formation (r1-r7 fragments) at an I-SceI DSB generated at the HIS3 locus. d, Fun30-Myc levels at MAT before and after HO induction measured by ChIP coupled to qPCR. Error bars define the s.e.m. of three independent experiments. e, 10-fold serial dilutions of yeast cultures.

Figure 3. SMARCAD1 promotes end resection, homologous recombination and cell survival after genotoxic insults in U2OS cells

a, Immunodetection (top) and quantification (lower right) of RPA foci 3 hr after 6 Gy of ionizing radiation. Western blot analysis of SMARCAD1 in cells transfected with individual or pooled siRNAs (lower left). Knockdown of Exo1 serves as a control. Nuclei with more than 10 RPA foci were scored. Error bars represent the s.e.m. of three independent experiments for all plots. b, Western blot analysis of SMARCAD1 (left) and quantification of homologous recombination frequencies using a DR-GFP assay (right). c, Clonogenic survival of SMARCAD1 knockdown cells treated with camptothecin or the PARP inhibitor ABT-888. d, Immunofluorescence staining of SMARCAD1 and γH2AX at DSBs induced by mCherry-LacI-FokI at a 256× LacO genomic array (top). Nuclease-deficient mCherry-LacI-FokI D450A was used as a control. Quantification of cells showing colocalization of SMARCAD1 and γH2AX at FokI-induced DSBs (bottom). e, Quantification of GFP-SMARCAD1, GFP-Exo1 and GFP-RPA accumulation at sites of laser micro-irradiation in live cells.

Figure 4. Model for Fun30/SMARCAD1 control of end resection through DSB-associated nucleosomes

Fun30/SMARCAD1 weaken histone-DNA interactions in nucleosomes flanking DSBs, which facilitates ssDNA production by the Exo1- and Sgs1/Top3/Rmi1 (STR)-Dna2 resection machineries. In the absence of Fun30/SMARCAD1 histone-DNA interactions limit the extent of resection, but plasmid-based overexpression of yeast or human Exo1 (pExo1), respectively, bypasses this impediment.