Activity of calf thymus DNA helicase E on cis-diamminedichloroplatinum (II)-damaged DNA.
Calf DNA helicase E (hel E) is a moderately processive, 3' to 5' helicase, active on nicked DNA, that we have proposed to have a role in DNA repair (Turchi, J. J., Murante, R. S., and Bambara, R. A. (1992) Nucleic Acids Res. 20, 6075-6080). Here we have examined its activity on a series of cis-diamminedichloroplatinum (II) (cis-DDP)-modified DNA substrates. Hel E was capable of efficiently displacing a primer strand containing, in an internal position, a cis-DDP-modified dGG. In a two-primer model system, calf DNA polymerase epsilon could successfully extend an upstream primer through a cis-DDP-modified down-stream primer, to the end of the complementary template strand, in a reaction dependent on hel E. However, the translocation of hel E was blocked by cis-DDP modification of the template strand. Primer displacement was completely prevented if the modified site was located just upstream of the primer. The DNA-dependent ATPase activity of helicase E was also reduced by cis-DDP modification of the template DNA. Substrate competition experiments indicated that cis-DDP-modified DNA templates did not sequester hel E. Substrate titration experiments suggested that there is a short delay without ATP hydrolysis before dissociation of helicase E from cis-DDP-modified template sites. Interestingly, hel E could displace a primer if the cis-DDP modification was on the template within the annealed region. Possible explanations for this are discussed. Taken together, these results are consistent with the proposal that hel E participates in DNA repair by displacing segments of damaged DNA.
Huang
L
L
Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, New York 14642.
Turchi
J J
JJ
Wahl
A F
AF
Bambara
R A
RA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002417:Cattle; D002945:Cisplatin / Q000378:metabolism*; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D018736:DNA Adducts; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair; D008969:Molecular Sequence Data; D013950:Thymus Gland / Q000201:enzymology*
D018736:DNA Adducts; D017931:DNA Primers; C057270:cisplatin-DNA adduct; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D002945:Cisplatin