Figure 4.

Mph1 DNA helicase activity on double flap-structured and equilibrating flap DNA. (A) Helicase assays were performed on double flap-structured substrates for various incubation periods (1, 4, 10 and 25 min). Two identical substrates but labeled on different oligonucleotides were used in these assays; one was labeled on the upstream oligonucleotide (lanes 1–6) and the other on the downstream oligonucleotide (lanes 7–12). (B) Helicase assays were performed on 5′-flap and double flap-structured substrates as a function of time. The amount of substrate unwound was plotted against the incubation period (0, 2, 5, 10 and 25 min). (C) Mph1 helicase assays were performed with equilibrating flap substrates using standard reaction conditions with increasing time of incubation (0, 2, 8, 25 and 60 min). Substrates were radio-labeled at the 5′-end of one of the two oligonucleotides annealed to the template as indicated with asterisks. (D) The mode of displacement of equilibrating flaps by Mph1 is illustrated.