Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1

Abstract

Repair of DNA double-strand breaks (DSBs) by homologous recombination requires resection of 5'-termini to generate 3'-single-strand DNA tails. Key components of this reaction are exonuclease 1 and the bifunctional endo/exonuclease, Mre11 (refs 2-4). Mre11 endonuclease activity is critical when DSB termini are blocked by bound protein--such as by the DNA end-joining complex, topoisomerases or the meiotic transesterase Spo11 (refs 7-13)--but a specific function for the Mre11 3'-5' exonuclease activity has remained elusive. Here we use Saccharomyces cerevisiae to reveal a role for the Mre11 exonuclease during the resection of Spo11-linked 5'-DNA termini in vivo. We show that the residual resection observed in Exo1-mutant cells is dependent on Mre11, and that both exonuclease activities are required for efficient DSB repair. Previous work has indicated that resection traverses unidirectionally. Using a combination of physical assays for 5'-end processing, our results indicate an alternative mechanism involving bidirectional resection. First, Mre11 nicks the strand to be resected up to 300 nucleotides from the 5'-terminus of the DSB--much further away than previously assumed. Second, this nick enables resection in a bidirectional manner, using Exo1 in the 5'-3' direction away from the DSB, and Mre11 in the 3'-5' direction towards the DSB end. Mre11 exonuclease activity also confers resistance to DNA damage in cycling cells, suggesting that Mre11-catalysed resection may be a general feature of various DNA repair pathways.

Figure 1. Mre11 and Exo1-dependent resection and repair of meiotic DSBs

a, Autoradiograph of Spo11-oligo formation (arrowheads) in Mre11 nuclease-defective cells: mre11-D56N, -H125N, -H59S, and in control spo11-Y135F cells where Spo11-DSBs do not form. Immunoprecipitated Spo11-oligo complexes are 3′ end-labelled using terminal transferase and separated on SDS-PAGE (see Fig. S1). Asterisk marks an unrelated labelling artifact; b-f, Timecourse of events during meiosis for the indicated genotypes: b-c, Quantitative analysis of DSB (b) and crossover (c) signals at HIS4::LEU2. DNA at each timepoint was digested with PstI (b) or XhoI (c), separated by agarose gel electrophoresis and blots hybridised with probes to LSB5 (b) or STE50 (c). DSB signals were quantified as a percentage of specific lane signal (see also Fig. S3); d, Relative extent of DSB resection at HIS4::LEU2 (at 4 hours in meiosis). DNA was digested with BglII and blots hybridised with a STE50 probe (see also Fig. S4); e, Western blot analysis of phosphorylated H2Ax and Hop1 (arrowhead) detected from TCA-precipitated whole cell lysates. Phosphorylated Hop1 indicated by dot. Asterisk marks cross-reacting band; f, Progression through anaphase I and II assessed by microscopic examination of DAPI-stained cells; g, Distribution of spore viabilities per tetrad (n; number of 4-spore tetrads dissected). The difference in distribution between mre11-H59S/exo1Δ and exo1Δ is highly significant (Chi-square test for goodness-of-fit; Chi value = 63.084, d.f. = 4, P < 0.0001). Absolute spore viabilities are: WT, 97%; mre11-H59S, 90%; exo1Δ, 76%, mre11-H59S/exo1Δ, 59%.

Figure 2. Mre11-exonucleolytic processing of DSB ends

a-b, Spo11-oligo detection in wildtype (WT) and mre11-H59S during meiosis. 3′ end-labelled Spo11 complexes are fractionated by SDS-PAGE (a) or by nucleotide resolution urea/PAGE after proteolytic removal of Spo11 peptide (b); c, Mre11-H59S displays reduced 3′-5′ exonuclease activity on a nicked duplex. Reactions were performed as for Fig. S2c. Stars indicate 5′ label, F3 3′-end has 5 nt extension and is refractory to Mre11-mediated resection; d, Chromatin association of Spo11-oligo complexes. Wildtype cell extracts were fractionated and the abundance of Spo11-oligo complexes assessed in soluble versus chromatin-enriched material; e-f, Nuclease resistance of Spo11-oligo complexes. Chromatin-enriched material from (d) was treated with DNase I, and abundance of Spo11-oligo complexes compared to the simultaneous degradation of genomic DNA (e), or of a control 55 nt oligonucleotide (f).

Figure 3. DNA damage sensitivity of exonuclease defective Mre11 cells

Ten-fold serial dilutions of the indicated strains were spotted onto solid media containing the indicated compounds and incubated at 30°C for 2 days (CPT) or 3 days (MMS).

Figure 4. Model for bidirectional processing of DSBs by Mre11 and Exo1

a, Following DSB formation with blocked ends (hatched squares), Mre11/Sae2-dependent nicks flanking the DSB ends create initiation sites for bidirectional resection by Exo1/Sgs1-Dna2 away from the DSB, and by Mre11 towards the DSB end. Such terminal blocks could arise after base damage, trapping of a topoisomerase, or by avid binding of the NHEJ complex. 3′ ends marked with triangles. Mre11/Sae2 may make multiple nicks on the 5′ strand (light grey arrows) facilitating resection; b, In meiosis, the DSB ends are terminally blocked by covalently bound Spo11 protein (grey ellipses), and may be protected from Mre11-dependent exonuclease degradation by a large metastable multisubunit complex (dashed outline), thereby generating the observed size-distribution of Spo11-oligonucleotide complexes.