Nickel effects on DNA polymerase activity in vitro
- NYU Medical Center, Tuxedo, NY (United States)
The effect of nickel ions (Ni{sup +2}) on DNA polymerase activity was studied in vitro using several different purified DNA polymerases and either primed single stranded or gapped duplex M13mp2 DNA as the template. The concentration of nickel giving 50% relative activity varied by several orders of magnitude for the different polymerases and for different templates. Under the conditions used, only E. coli Polymerase I-Klenow fragment (Pol I-KF) was able to effectively use Ni{sup +2} as the activation cation in place of Mg{sup +2}. Besides inhibiting nucleotide incorporation, nickel also inhibited primer extension by T7 and T4 polymerases. Nickel was significantly more inhibitory to Sequenase-2.0, an exo minus derivative of T7 polymerase, than to T7 polymerase itself. It also preferentially inhibited the 3{prime}-5{prime} exonuclease activity of T7 polymerase. This may indicate that Ni{sup +2} binds preferentially to the exonuclease active site of T7 polymerase. A rapid, minus dNTP, primer-extension assay for polymerase fidelity was also performed in the presence of Ni{sup +2}. In the absence of one of the 4 required dNTPs an accurate polymerase will pause at or before the first base that pairs with the missing dNTP. An inaccurate polymerase will misincorporate an incorrect base and bypass this and subsequent pause sites. Using this assay nickel did not cause misincorporation by AMV polymerase. However, the presence of increasing Ni{sup +2} (from 10 to 200 {mu}M) first increased and then decreased the fidelity of Pol I KF and exo minus KF and decreased the fidelity of Sequenase. The fidelity of T7 polymerase was not altered by Ni{sup +2}, despite an almost complete inhibition of the 3{prime}-5{prime} exonuclease activity by high Ni{sup +2} concentrations. These results indicate that nickel mutagenesis may vary significantly depending on the polymerase.
- OSTI ID:
- 88918
- Report Number(s):
- CONF-9405324-; ISSN 0893-6692; CNN: Grant CA45664; Grant ES00260; TRN: 95:004220-0063
- Journal Information:
- Environmental and Molecular Mutagenesis, Vol. 23, Issue Suppl.23; Conference: 25. annual meeting of the Environmental Mutagen Society, Portland, OR (United States), 7-12 May 1994; Other Information: PBD: 1994
- Country of Publication:
- United States
- Language:
- English
Similar Records
The effects of chromium(III) on DNA replication across O{sup 6}. Methylguanine by DNA polymerase {beta} and E. coli DNA polymerase I-Klenow fragment
Kinetic mechanism of DNA polymerase I