Biochemistry 1985, 24, 5810-5817
5810
On the Fidelity of DNA Replication: Manganese Mutagenesis in Vitro? Robert A. Beckman,* Albert S.Mildvan,$ and Lawrence A. Loeb*J Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 981 95, and Department of Physiological Chemistry, Johns Hopkins Medical School, Baltimore, Maryland 21 205 Received January 17, 1985
Manganese is mutagenic in vivo and in vitro in studies with a variety of enzymes and templates. Using Escherichia coli D N A polymerase I with poly[d(A-T)] and 4 x 1 7 4 D N A templates, we analyzed the mechanism of manganese mutagenesis by determining the dependence of error rate on free Mn2+ concentration and comparing this to measured dissociation constants of Mn2+ from enzyme, template, and deoxynucleoside triphosphate substrates. This comparison suggests several conclusions: (1) At very low Mn2+ concentrations, the enzyme is activated a t high fidelity. Thus, it is unlikely that activation with manganese per se significantly alters the conformation of the enzyme so as to affect nucleotide selection. (2) At low free Mn2+ concentrations (
