FIGURE 6.
Dna2 rescues RPA-inhibited ligation of long flaps. A, cleavage by FEN1 (5 fmol) and Dna2 (50 fmol) and ligation by LigI (25 fmol) were assayed on the pre-created flap substrate (U2:T2:D3) in the presence of various combinations of pol δ (23 fmol), PCNA (25 fmol), RFC (25 fmol), and RPA (200 fmol) as indicated in the figure and as described under “Experimental Procedures.” B, synthesis by pol δ (23 fmol) and ligation by LigI (25 fmol) were assayed on unlabeled extended pre-created flap substrate (U2:T2:D4) in the presence of 0.5 μm [α-32P]dGTP and various combinations of PCNA (25 fmol), RFC (25 fmol), FEN1 (5 fmol), Dna2 (50 fmol), and RPA (200 fmol) as indicated in the figure and as described under “Experimental Procedures.” Lane 1 contains upstream primer (U2) radiolabeled at the 5′-end. Substrate depictions and designations are as in Fig. 1.