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The Effect of Divalent Nickel (Ni²⁺) on in Vitro DNA Replication by DNA Polymerase ¹

Department of Environmental Medicine, New York University Medical Center, Tuxedo, New York 10987

The effects of the carcinogenic metal nickel on DNA polymerase (pol ) 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 as monitored by [³H]dTMP incorporation into an activated DNA template. The apparent K_m for Ni²⁺-dependent pol incorporation of dTTP was estimated to be 25 µM, which was about 10 times higher than the K_m for Mg²⁺ (2.3 µM). Above 0.25 mM, Ni²⁺ caused a dose-dependent inhibition of pol activity and the K_i was calculated to be 1.5 mM. Scatchard analyses showed that Ni²⁺ binds to affinity-purified pol and associated proteins at two tight binding sites with a K_d of 50 µM and at eight weak binding sites with a K_d of 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.

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.

Received 11/22/93. Accepted 3/ 3/94.