DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme.
We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound.
Thömmes
P
P
Department of Pharmacology and Biochemistry, University of Zürich-Irchel, Switzerland.
Hübscher
U
U
D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D009687:Nuclear Proteins / Q000302:isolation & purification; D018809:Proliferating Cell Nuclear Antigen; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
D006882:Hydroxyapatites; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D017886:Durapatite; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases