Human DNA2 preferentially degrades branched DNA in a 5′-3′ direction in reactions stimulated by WRN. (A) Degradation of a four-way junction by human DNA2 (hDNA2) in the presence of hRPA (native 6% polyacrylamide gel) (B) Experiment as in A, but with dsDNA. (C) Quantitation of data from A and B. Averages shown ± SEM; n = 2. (D) DNA degradation is stimulated by hRPA. The data points from +hRPA condition are the same as in C. Averages shown ± SEM; n = 2. (E) Quantitation of degradation of a 3′ or 5′ ssDNA-tailed three-way junction by hDNA2. The reactions were performed in 3 mM magnesium acetate and 22.3 nM hRPA. Averages shown ± SEM; n = 2. (F) Kinetics of degradation of a four-way junction by hDNA2 (9 nM) in the presence of hRPA (denaturing 20% polyacrylamide gel). The substrate was labeled at the 5′ end (*). D294A, nuclease-dead variant of hDNA2. (G) Experiment as in F, but using a four-way junction labeled at the 3′ end. (H) Quantitation of DNA cleavage near (less than 15 nt) a 5′ or 3′ DNA end from experiments of F and G. Averages shown ± SEM; n = 2. (I) WRN and hDNA2 degrade four-way junction DNA in a synergistic manner. Reactions with indicated hDNA2 and/or WRN concentrations and 65 nM hRPA were analyzed on a 6% native polyacrylamide gel. Heat, partially heated DNA substrate indicating the positions of DNA unwinding intermediates. (J) Quantitation of four-way junction and dsDNA degradation by human EXO1 (hEXO1). Averages shown ± SEM; n = 2.