Figure 1.

BLM and DNA2 resect dsDNA. (A) Resection of plasmid-length dsDNA. Nuclease reactions were performed using 5′- or 3′-end-labeled 2.7-kb DNA; reactions contained 400 nM RPA and 2 mM MgCl₂. (Lane Δ) Heat-denatured substrate. (Lanes 1,6) Substrate. (Lanes 2,7) BLM. (Lanes 3,8) BLM and 2 nM DNA2. (Lanes 4,9) BLM and 4 nM DNA2. (Lanes 5,10) DNA2 (4 nM). Single plus sign (+) and double plus signs (++) refer to 2 nM and 4 nM DNA2, respectively. The positions of the intact substrate (2.7 kb), unwound substrate (ssDNA), resection products, and molecular size markers are indicated. (B) DNA2 cleaves forked DNA with 5′ → 3′ polarity. Nuclease reactions with varying DNA2 concentrations (1, 2, and 4 nM) were performed using 5′- or 3′-end-labeled 50-bp forked DNA (1.5 nM ends, 75 nM nucleotides). Reactions contained 10 nM RPA and 5 mM MgCl₂. (Lane 1) Substrate. (Lanes 2–4) Reactions with 5′-end-labeled fork. (Lanes 5–7) Reactions with 3′-end-labeled fork. (Lane Δ) Heat-denatured substrate. (C) RPA modulates DNA2-mediated cleavage of forked DNA. Nuclease reactions with DNA2 (0, 1, 2, and 4 nM) were performed using 5′-end-labeled 50-bp forked DNA in the absence or presence of indicated ssDNA-binding protein (RPA or E. coli SSB). All reactions contained 5 mM MgCl₂. (Lanes 1–4) DNA2 alone. (Lanes 5–8) DNA2 and SSB. (Lanes 9–12) DNA2 and RPA. The positions of the intact fork, unwound substrate, and cleavage products are indicated schematically.