Figure 2.

BLM and DNA2 interact specifically to resect dsDNA. (A) BLM-mediated unwinding is essential for resection by BLM and DNA2. Nuclease reactions were performed using a 5′-end-labeled 50-bp DNA fragment. Where indicated, wild-type BLM (WT) was substituted with helicase-dead K695R mutant (HD) or E. coli RecQ. Reactions contained 10 nM RPA and 5 mM MgCl₂. (Lane 1) Substrate. (Lanes 2–4,5–7) BLM (wild type) (5, 10, and 20 nM) in the absence or presence of DNA2, respectively. (Lanes 8–10,11–13) BLM (helicase-dead) (20, 50, and 100 nM) in the absence or presence of DNA2, respectively. (Lane Δ) Heat-denatured substrate. (Lanes 14–16,17–19) RecQ (20, 50, and 100 nM) in the absence or presence of DNA2, respectively. (Lane 20) DNA2 alone. (B) The percentage of dsDNA unwound or resected, from experiments as shown in A, plotted as a function of helicase concentration. The percentage resected or unwound was obtained by expressing the amount of DNA degraded or unwound, respectively, as a percentage of total signal, and is plotted as “products.” Error bars indicate standard deviation from three to five independent experiments and are smaller than the symbols when not evident. (C) Resection requires the nuclease but not helicase activity of DNA2. Reactions were performed as described in A, with the exception that, where indicated, DNA2 was substituted with helicase-dead (HD) or nuclease-dead (ND) DNA2. (Lane 1) BLM. (Lanes 2–4) BLM with wild-type (WT), K671E (HD), or D294A (ND) DNA2, respectively. (Lanes 5–7) Wild type (WT), helicase-dead (HD), and nuclease-dead (ND) DNA2, respectively. (Lane Δ) Heat-denatured substrate. (D) Sgs1 can substitute for BLM at low [Mg²⁺] (2 mM). Where indicated, BLM was replaced with Sgs1.The concentration of Mg²⁺ was as indicated in the figure. (Lane 1) Substrate. (Lanes 2,3) Reactions with BLM (absence or presence of DNA2). (Lanes 4–9) Reactions with Sgs1 (absence or presence of DNA2). (Lane Δ) Heat-denatured substrate. (E) BLM and DNA2 interact directly. Pull-down experiments were performed with BLM and DNA2 in three sets: BLM alone, BLM and DNA2, and DNA2 alone. The bound fractions were analyzed by gel electrophoresis followed by Sypro Orange staining. (Lanes 1,2) Approximately 250 ng of BLM and DNA2, respectively. (Lanes 3–5) Pull-downs with BLM alone, BLM and DNA2, and DNA2 alone, respectively. (Lanes 6–8) Same as lanes 3–5, but in the presence of 12.5 U of benzonase. The positions of size markers (in kilodaltons), BLM, and DNA2 are indicated. (F) Yeast Dna2 functions with BLM in DNA end resection. Reactions were performed as described in A, except that, when indicated, DNA2 was substituted with Dna2 (human RPA throughout). (Lane 1) Substrate. (Lane 2) BLM. (Lane 3) BLM–DNA2. (Lane 4) BLM–Dna2. (Lane 5) DNA2. (Lane 6) Dna2. DNA2 and Dna2 are indicated as “H” and “Y,” respectively.