Mammalian DNA helicase

Abstract

A forked DNA was constructed to serve as a substrate for DNA helicases. It contains features closely resembling a natural replication fork. The DNA was prepared in large amounts and was used to assay displacement activity during isolation from calf thymus DNA polymerases a holoenzyme. One form of DNA polymerase α holoenzyme is possibly involved leading strand replication at the replication fork and possesses DNA dependent ATPase activity (Ottiger, H.-P. and Hüibscher, U. (1984) Proc. Natl. Acad. Sc1. USA 81, 3993-3997). The enzyme can be separated from DNA polymerase a by velocity sedimentation 1n conditions of very low 1onic strength and then be purified by chromatography on Sephacryl S-200 and ATP-agarose. At all stages of purification, DNA dependent ATPase and displacement activity profiles were virtually superimposable. The DNA dependent ATPase can displace a hybridized DNA fragment with a short single-stranded tail at its 3′hydroxyl end only 1n the presence of ATP, and this displacement relies on ATP hydrolysis. Furthermore, homogeneous single-stranded binding proteins from calf thymus as well as from other tissues cannot perform this displacement reaction. By all this token the DNA dependent ATPase appears to be a DNA helicase. It 1s suggested that this DNA helicase might act in concert with DNA polymerase α at the leading strand, possibly pushing the replication fork ahead of the polymerase.