Mammalian DNA helicase

• Published in: Nucleic acids research Vol. 13; no. 15; pp. 5471 - 5483
• Main Authors: Hübscher, Ulrich, Stalder, Hans-Peter
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 12.08.1985
• Subjects: Adenosine Triphosphatases - isolation & purification; Adenosine Triphosphatases - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; calf thymus; Cattle; Centrifugation, Density Gradient; Chromatography; DNA biosynthesis; DNA helicase; DNA Helicases; DNA polymerase I; DNA Polymerase II - metabolism; DNA Replication; DNA-Binding Proteins - metabolism; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; replication forks; T-Phages - enzymology; Purification; Holoenzyme; Vertebrata; DNA-dependent ATPase; Mammalia; DNA polymerase α; DNA; Substrate specificity; Molecular association; Reaction mechanism; Isolation; Replication; Unwinding enzyme
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/13.15.5471

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.