DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2

Abstract

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3'-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5'-terminated strand of the DNA break and to inhibit 3' to 5' degradation by Dna2, actions that generate and protect the 3'-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11-Rad50-Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3-Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes.

Figure 1. Sgs1 and Dna2 resect DNA in a reaction dependent on yeast RPA

a, Purified Dna2 (80 ng) and Sgs1 (880 ng) stained with Coomassie Brilliant Blue. b, Linear pUC19 dsDNA incubated with Sgs1 and/or Dna2, and RPA (3 μM); “SSB”: SSB substituted for RPA; “Heat”: heat-denatured dsDNA; ”annealed DNA”: partial unwinding and annealing of DNA. c, and d, Quantification of experiments as shown in b. e, Dna2 and Sgs1 physically interact in the absence or presence of RPA (lanes 4 and 5). f, Resection by Dna2 (1 nM) is specific for yeast Sgs1 helicase; RPA is 3 μM.

Figure 2. Sgs1, Dna2, and RPA preferentially resect the 5'-terminated strand of a DNA break

a, Assay. b, Annealing of oligonucleotide to resection products (0.5 nM Sgs1, 0.5 nM Dna2, and 3 μM RPA) using probe complementary to either top (3'-) or bottom (5'-) strand, 300 nucleotides from end. c, Quantification of DNA end resection at various distances from DSB, based on experiments as shown in b.

Figure 3. RPA promotes 5'→3' degradation by Dna2 and inhibits 3'→5' degradation

a, Duplex DNA substrates containing either a 5'- or 3'-ssDNA flap (red asterisk indicates the ³²P-label) incubated with Dna2 (15 nM) and indicated RPA. b, Illustration summarizing results from panel a and Supplementary Figs. 4 and 5 showing modulation of ssDNA nuclease activities of Dna2.

Figure 4. Top3-Rmi1 and MRX complexes stimulate DNA resection by Dna2-Sgs1-RPA

a, Resection kinetics: Sgs1 (0.3 nM), Dna2 (1 nM), RPA (3 μM), and Top3-Rmi1 heterodimer (10 nM). b, Top3-Rmi1-dependent stimulation of DNA resection: Sgs1 (0.5 nM), Dna2 (0.5 nM) and RPA (3 μM). c, Resection kinetics in high salt buffer (5 mM Mg²⁺ and 100 mM Na⁺): Sgs1 (7.5 nM), Dna2 (1 nM), RPA (3 μM ), and Top3-Rmi1 heterodimer (15 nM). d, Quantification of experiments as in c. e, DNA unwinding in high salt buffer; RPA (3 μM). f, Resection in high salt buffer; RPA (3 μM). g, Stimulation of resection in high salt buffer by Top3-Rmi1 (15 nM) and MRX (20 nM) using Dna2 (1 nM), RPA (3 μM), Sgs1 (0, 1.3, 1.7, 2.5, 3.8, 5.7 and 8.6 nM). h, Quantification of experiments as in g. i, Unwinding of Y-structure DNA in high salt buffer: Sgs1 (200 pM); RPA (2.25 nM); and MRX (5 nM).