Figure 4
Mutual regulation of the nuclease activities of Hef and HAN. (a) The structure of the substrate is shown with the sites labeled by FITC and Cy5. The site cleaved by Hef is indicated by arrowhead. (b) The effects of HAN and Hef on each other on the fork-structured DNA. Hef (5 nM as the dimer) and HAN (100 nM) were incubated with 50 nM of each substrate at 50 °C for 2, 5, 10, 20, and 30 min. The reaction products were analyzed by 8 M urea-15% PAGE. The band assignments are indicated on the side of the panel: s, substrates; p, cleaved products by HAN (p-HAN, Cy5-labeled DNA) and Hef (p-Hef, FITC-labeled DNA). (c) Quantification of Hef nuclease activity in the presence or absence of HAN. The FITC-labeled cleavage products generated by Hef in panel b were quantified and represented as percentage of the total amount of substrate DNA. The SEM was calculated from three independent experiments. (d) Quantification of HAN nuclease activity in the presence or absence of Hef. The Cy5-labeled products generated by HAN in panel b were quantified and plotted. The SEM was calculated from three independent experiments.
