DNA-dependent adenosine triphosphatase B from mouse FM3A cells has DNA helicase activity

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Abstract
We have detected at least four forms of DNA-dependent ATPase in mouse FM3A cell extracts [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533]. The purified fraction of one of the four forms, ATPase B, has been shown to have DNA helicase activity by using a DNA substrate which permits the detection of limited unwinding of the helix. The DNA substrate consists of single-stranded circular fd DNA and the hexadecamer complementary to the fd DNA, which bears an oligo (dT) tail at the 3'' terminus. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.5 S on glycerol gradient centrifugation. The helicase required a divalent cation for activity (Mg2+ .simeq. Mn2+ > Ca2+). The optimal concentrations of these divalent cations were 5 mM. The requirement of divalent cations of the DNA helicase activity was very similar to that for the DNA-dependent ATPase activity of ATPase B. The helicase activity was absolutely dependent on the presence of a nucleoside triphosphate. ATP was the most effective cofactor among the ribo- and deoxyribonucleoside triphosphates tested, and considerable levels of helicase activity were observed with other ribo- and deoxyribonucleoside triphosphates. The efficiency of a nucleoside triphosphate to serve as cofactor for the helicase acitvity correlated with the capacity of the nucleotide to serve as substrate for the DNA-dependent ATPase activity. The nonhydrolyzable ATP analogues such as adenosine 5''-0-(3-thiotriphosphate) were not effective for the helicase activity. The helicase displaced the hexadecamer with no tail as well as the hexadecamer bearing the 3'' or 5'' tail. The efficiency of displacement was almost the same among the three substrates.