DNA helicases associated with DNA polymerases from human cells.
A crude DNA polymerase fraction partially purified from a low salt extract of HeLa cells was fractionated on a hydroxylapatite column by an elution with a linear gradient of potassium phosphate. By this procedure, DNA polymerase alpha, delta and epsilon were separated from each other. DNA helicase activities were detected in the DNA polymerase alpha and delta fractions but not in the epsilon fraction. Characterization of DNA helicases after further purification on heparin column revealed that the DNA helicase in the DNA polymerase alpha fraction required ATP (or dCTP) in addition to ATP (or dATP). Both DNA helicases translocated on single-stranded DNA in the same direction of 3' to 5'. By a repeated gel-filtration on Superose 6 (SMART system), activities of DNA polymerase alpha and delta were eluted at positions of approx. 600 kDa and 400 kDa, respectively, and the activities of DNA helicases were well associated with those of corresponding DNA polymerases. These results strongly suggest that the DNA helicases described here are physically associated with DNA polymerase alpha and delta to make large complexes.
Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Japan.
T
T
Sakurai
M
M
Suzuki
S
S
Yoshida
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004257:DNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data
D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases