A novel DNA helicase from calf thymus.
We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.
Siegal
G
G
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Turchi
J J
JJ
Jessee
C B
CB
Myers
T W
TW
Bambara
R A
RA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D004249:DNA Damage; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016622:Silver Staining; D013950:Thymus Gland / Q000201:enzymology*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases