Figure 3.
Changes in Enzymatic Activities of Altered Forms of DNA2
(A) Endonuclease activities of WT and altered forms of DNA2. In the top panel is the DNA double-flap substrate. WT, p.Arg284His, p.Lys313Glu, or p.Val723Ile human DNA2 (0.8 nM) was incubated with 0.25 pmol 5′ end 32P-labeled flap DNA substrate. Reactions were carried out at 37°C for 10, 20, 40, and 60 min. Arrows indicate the cleavage sites. In the bottom panel is the quantification of DNA2 endonuclease activity. Error bars indicate SEM.
(B) Helicase activities of WT and altered forms of DNA2. In the top panel is the DNA helicase substrate. DNA2 was first altered for the elimination of nuclease activity (p.Asp294Ala) so that helicase activity could be assayed. A helicase mixture containing 1 fmol 3′-end-labeled duplex substrate and 100 nM purified recombinant p.Asp294Ala, p.Asp294Ala/p.Arg284His, p.Asp294Ala/p.Lys313Glu, or p.Asp294Ala/p.Val723Ile DNA2 was incubated at 37°C for 10, 20, 40, and 60 min. In the bottom panel is the quantification of DNA2 helicase activity. Error bars indicate SEM.
(C) ATPase activity of WT and altered forms of DNA2. ATPase activity of WT, p.Arg284His, p.Lys313Glu, or p.Val723Ile forms of human DNA2 was measured with an ATPase assay kit. A reaction mixture containing 30 nM recombinant protein and 1 μg ssDNA was incubated at 37°C for 10, 20, 40, and 60 min. In each panel, the p value was calculated by a Student’s t test. Error bars indicate SEM.