Figure 7.

Pif1-catalyzed annealing of partial duplex substrates with 3′-overhangs. (a) Schematic illustration for strand annealing experiments using two substrates with 30bp duplexes and complementary 30nt overhangs to generate a 90bp blunt end dsDNA product. (b) Shown are results for Pif1 (200 nM) annealing of 30nt-30bp (2.6 nM) and radiolabeled 30bp-30nt (2 nM) to generate a 90bp blunt end DNA product in the presence of ATP and MgCl₂ (circles). Pif1 annealing in the presence of 200 nM Rim1 (squares) and 200 nM RPA (diamonds) was also measured. Annealing activity of RPA alone is shown as empty circles, Rim1 alone is shown as X. Spontaneous annealing in the absence of enzyme was also measured (triangles). Data were fit to a simple annealing mechanism (Scheme 2) to obtain second-order rate constants for annealing by Pif1 (9.2 × 10⁶ M^–1 s^–1), Pif1 and RPA (1.5 × 10⁷ M^–1 s^–1), Pif1 and Rim1 (4.8 × 10⁶ M^–1 s^–1), and RPA (1.2 × 10⁷ M^–1 s^–1). (c) Schematic illustration for strand annealing experiments using two substrates with 30bp duplexes and 30nt overhangs containing 4nt of mismatch in the middle of otherwise complementary sequences to generate a 90bp blunt end dsDNA product containing a 4nt bubble. (d) Results for Pif1 (200 nM)-catalyzed annealing of 30nt-30bp-mut (2.6 nM) and radiolabeled 30bp-30nt (2 nM) to generate a 90bp blunt end DNA product containing a 4nt bubble in the presence of ATP and MgCl₂ (circles). Spontaneous annealing in the absence of enzyme was also measured (triangles). Data were fit to a simple annealing mechanism (Scheme 2) to obtain a second-order rate constant for annealing by Pif1 (4.1 × 10⁶ M^–1 s^–1).