Abstract

The effects of the carcinogenic metal nickel on DNA polymerase (pol ) activity and fidelity have been analyzed. In the absence of Mg 2+ , the presence of Ni 2+ ions at concentrations below 0.25 m M gave rise to a dose-dependent activation of pol as monitored by 3 HdTMP incorporation into an activated DNA template. The apparent K m for Ni 2+ -dependent pol incorporation of dTTP was estimated to be 25 µ M , which was about 10 times higher than the K m for Mg 2+ (2.3 µ M ). Above 0.25 m M , Ni 2+ caused a dose-dependent inhibition of pol activity and the K i was calculated to be 1.5 m M . Scatchard analyses showed that Ni 2+ binds to affinity-purified pol and associated proteins at two tight binding sites with a K d of 50 µ M and at eight weak binding sites with a K d of 4 m M . In the presence of 2 m M Mg 2+ , the addition of Ni 2+ 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 Ni 2+ concentration. Lastly, Ni 2+ increased the incorporation of the modified nucleotide dideoxy-CMP in reactions using varying ratios of dideoxy-CTP/dCTP. 1 This study was supported by National Institute of Environmental Health Sciences Grants ES-00260 and ES-04895 and by United States Environmental Protection Agency Grant R-184751. E. T. S. was supported by NIH Grants CA-46554 and ES-06498. 2 To whom requests for reprints should be addressed, at New York University Medical Center, Nelson Institute of Environmental Medicine, Long Meadow Road, Tuxedo, NY 10987.