A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1-Dna2 in long-range DNA end resection

Abstract

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.

Keywords: DNA binding protein; DNA damage; DNA end resection; DNA gap; DNA helicase; DNA nick; DNA repair; Exo1; Sgs1-Dna2; homologous recombination; nuclease.

Figure 1.

The inhibitory effect of Ku on Exo1- or Dna2-mediated long-range resection is relieved by a nick. A, schematic of DNA substrate preparation (see “Experimental procedures” for details). The asterisk denotes the ³²P label. B, activity of Exo1 (8 nm) on linear dsDNA without or with a nick (1 nm) prebound by Ku (8, 16, 32, and 64 nm). The results from three independent experiments were graphed with the error bars representing S.D. C, the activity of Dna2 (32 nm) on dsDNA or nick-containing dsDNA substrate prebound by Ku as in B was tested with Sgs1 (16 nm) and RPA (800 nm). The results were graphed as in B. See also Fig. S1.

Figure 2.

Nuclease activity of Exo1 and Dna2–Sgs1–RPA on circular dsDNA with a nick. A, reaction schematic. The asterisk denotes the ³²P label in the substrate. B, the circular nick-containing substrate (1 nm) was incubated with Exo1 (0.25 nm) in the presence of RPA (800 nm) and/or MRX (16 nm) for the indicated times. The results from three independent experiments were graphed with the error bars representing S.D. C, the activity of Dna2 (8 nm) was tested with combinations of Sgs1 (8 nm), RPA (800 nm), TR (8 nm), and MRX (16 nm) as indicated. The results were graphed as in B. See also Fig. S2.

Figure 3.

Resection by Exo1 or Dna2–Sgs1–RPA initiated at a DNA nick occurs in the 5′ to 3′ direction. A, schematic of reaction and Southern blot analysis. P500 and P2500 are 20-nt probes that correspond to DNA segments in the nicked DNA strand 500 and 2500 nt from the 5′ terminus of the nick, respectively. The asterisk denotes the ³²P label in the probes. B, the DNA nick-containing substrate (3 nm) was incubated with Exo1 (0.75 nm) for the indicated times and analyzed using the P500 or P2500 probe. C, resection mediated by Dna2 (24 nm) in conjunction with Sgs1 (24 nm) and RPA (2400 nm) was analyzed as in B. The red arrows denote ssDNA region exposed by 5′ strand resection at the early time points. D, probes used in the following experiments. P2500R is a 20-nt probe that is complementary to the indicated locale of the nicked DNA strand in the substrate. The asterisk denotes the ³²P label in the probes. E, analysis of the nicked circular DNA substrate with or without heat denaturation (HD) or digestion with T7 exonuclease (Exo) by agarose gel electrophoresis and ethidium bromide staining. F, the nicked circular DNA substrate was incubated with T7 exonuclease, Exo1, or Sgs1–Dna2–RPA followed by hybridization with radiolabeled probe P2500 or P2500R.

Figure 4.

Activity of Dna2 and Exo1 on circular ssDNA. A, Dna2 or Exo1 was incubated with circular ssDNA φX174 (10 nm) in the absence or presence of RPA (8 μm). B, the results from three independent experiments (A) were graphed with the error bars representing S.D.

Figure 5.

Sgs1-mediated unwinding of circular dsDNA substrates with a nick or gap. A, reaction schematic involving the use of a nick-containing circular DNA substrate. The asterisk denotes the ³²P label in the substrate. B, Sgs1 (1, 2, 4, 8, 16, and 32 nm) was tested on the nick-containing DNA substrate (1 nm) in the presence of RPA (800 nm). The results from three independent experiments were graphed with the error bars representing S.D. HD, heat denaturation. C, yeast RPA, hRPA, human SOSS1 and SOSS2, and E. coli SSB (800 nm each) were tested for their effect on unwinding of the nick-containing substrate by Sgs1 (32 nm) as in B. The results were graphed as in B. D, TR (4 nm) and MRX (8 nm) were tested, alone or in combination, for their effect on unwinding of the nick-containing substrate by Sgs1 (32 nm) and RPA (800 nm) as in B. The results were graphed as in B. E, the nick-containing or gapped circular dsDNA (0.5 nm each) was incubated with Sgs1 (8, 16, and 32 nm) and RPA (400 nm). The asterisk denotes the ³²P label in the substrate. See also Fig. S3.

Figure 6.

Exo1- or Dna2-mediated 5′ strand resection from an entry site created by MRX–Sae2. Ku, while protecting the DNA end from exonucleolytic digestion by Exo1, promotes 5′ strand endonucleolytic cleavage by MRX–Sae2. The resulting nick can serve as an entry site for Exo1 (A) or Sgs1–TR–Dna2–RPA (B) to carry out long-range DNA end resection in the 5′–3′ direction.