Fig 1.

Kinetics of D-loop unwinding activity of UvsW helicase and the effect of gp32 on its unwinding activity. (A) Schematic representation of RecA-mediated generation of the D-loop DNA substrate and UvsW-mediated unwinding reaction of the D-loop substrate. The 80 nt invasion primer was ³²P-radiolabeled at the 5′-end using T4 PNK and incubated with RecA and ATP for 5 min at 37C. The D-loop substrate generated was purified as described in Experimental section. The D-loop unwinding reactions were carried out by incubating the reaction mixture containing ATP (5 mM) and UvsW (200 nM) alone or in the presence of gp32 proteins (2 mM). (B) Products of D-loop unwinding reaction mediated by UvsW at regular time intervals resolved on an agarose gel (lanes 1-6). Unwound primers and intact D-loop substrates are indicated on the gel. The reactions that included gp32 (lanes 7-12) and gp32-A (lanes 13-18) are also shown. (C) The unwinding reactions in (B) were quantified using ImageQuant software and the time traces for the drop in the signal of the reactant is shown for reactions mediated by UvsW, UvsW/gp32 and UvsW/gp32-A are shown in red, blue and green respectively.