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RPA inhibits processing and ligation of long flaps in the absence of Dna2. Ligation by LigI (25 fmol) was 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), FEN1 (20 fmol), RPA (200 fmol), and varying amounts of Pif1 (25, 50, or 100 fmol) as indicated in the figure and as described under “Experimental Procedures.” Substrate depiction and designations are as in Fig. 1.
