DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes.
Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).
Tuteja
N
N
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Rahman
K
K
Tuteja
R
R
Ochem
A
A
Skopac
D
D
Falaschi
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology*; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D004265:DNA Helicases