Figure 4.

The influence of one flap on the hDna2-catalyzed cleavage of the other flap in a two-flapped substrate. The four partial duplex substrates used are shown at the top of figure (* indicates ³²P-labeled end). The 5′-ssDNA tail and 3′-ssDNA consist of homopolymer dT. The number in the schematic diagram of substrates used indicates the length of ssDNA tail. In case of the fork substrates, the length of 5′-ssDNA and 3′-ssDNA are identical. (A) Each substrate was incubated with increasing amounts of hDna2 at 37°C for 10 min and the reaction products formed were analyzed on 20% denaturing polyacrylamide gel. The numbers shown on the right of the figure indicate the size of the markers. (B) Quantitation of the results obtained in (A). The double-flap substrates used are shown at the top of figure (* indicates ³²P-labeled end). n = number indicates length of 5′ or 3′ ssDNA in double-flap substrate. B denotes the boiled substrate. The numbers at the bottom of figure indicate fmol of nucleolytic products. (C) The double-flap substrates containing a 30 nt 5′ flap and a 3′ flap of various lengths (0, 5, 10 or 30 nt) were prepared. Each substrate was incubated with increasing amounts of hDna2 at 37°C for 10 min. The products formed were analyzed as described under Materials and Methods. (D) The double-flap substrates containing a 30 nt 3′ flap and a 5′ flap of various length (0, 6, 13 or 30 nt) were prepared. Each substrate was incubated with increasing levels of hDna2 at 37°C for 10 min. The reaction products formed were analyzed as described under Materials and Methods.