title: The effect of divalent nickel (Ni2+) on in vitro DNA replication by DNA polymerase alpha.
abstract: The effects of the carcinogenic metal nickel on DNA polymerase alpha (pol alpha) activity and fidelity have been analyzed. In the absence of Mg2+, the presence of Ni2+ ions at concentrations below 0.25 mM gave rise to a dose-dependent activation of pol alpha as monitored by [3H]dTMP incorporation into an activated DNA template. The apparent Km for Ni(2+)-dependent pol alpha incorporation of dTTP was estimated to be 25 microM, which was about 10 times higher than the Km for Mg2+ (2.3 microM). Above 0.25 mM, Ni2+ caused a dose-dependent inhibition of pol alpha activity and the Ki was calculated to be 1.5 mM. Scatchard analyses showed that Ni2+ binds to affinity-purified pol alpha and associated proteins at two tight binding sites with a Kd of approximately 50 microM and at eight weak binding sites with a Kd of approximately 4 mM. In the presence of 2 mM Mg2+, the addition of Ni2+ to the reactions caused an inhibition of polymerase activity. The inhibition patterns tended to switch from competitive to mixed-type to noncompetitive as a function of Ni2+ concentration. Lastly, Ni2+ increased the incorporation of the modified nucleotide dideoxy-CMP in reactions using varying ratios of dideoxy-CTP/dCTP.
authors.affiliation: Department of Environmental Medicine, New York University Medical Center, Tuxedo 10987.
authors.forename: Y E
authors.initials: YE
authors.lastname: Chin
authors.forename: E T
authors.initials: ET
authors.lastname: Snow
authors.forename: M D
authors.initials: MD
authors.lastname: Cohen
authors.forename: N T
authors.initials: NT
authors.lastname: Christie
mesh_terms: D003570:Cytidine Triphosphate / Q000378:metabolism; D004257:DNA Polymerase II / Q000187:drug effects / Q000378:metabolism*; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D009532:Nickel / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D003570:Cytidine Triphosphate; D009532:Nickel; D004257:DNA Polymerase II; D008274:Magnesium