Purification and characterization of a DNA helicase from Saccharomyces cerevisiae.
A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation. The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis. scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM. The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min. This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min. The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound. The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used. The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl.
Bean
D W
DW
Department of Biology, University of North Carolina, Chapel Hill 27599.
Kallam
W E
WE
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases