DNA helicase IV from HeLa cells.
Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized. The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA. From 440g of HeLa cells we obtained 0.31 mg of pure protein. Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities. The apparent molecular weight is 100 kDa. It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP. The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly [G] (as phosphate). The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res. 18, 6785-6792, 1990). It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure. Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid.
Tuteja
N
N
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Rahman
K
K
Tuteja
R
R
Falaschi
A
A
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins; D004492:Edetic Acid / Q000494:pharmacology; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011068:Poly G / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D002412:Cations; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D015636:Magnesium Chloride; D011068:Poly G; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D014357:Trypsin; D000251:Adenosine Triphosphatases; C059716:helD protein, E coli; D004265:DNA Helicases