Influence of divalent metal activator on the specificity of misincorporation during DNA synthesis catalyzed by DNA polymerase I of Escherichia coli

To test whether the identity of divalent metal activator affects the specifity of misincorporation during polymerization catalyzed by E. coli DNA polymerase I, we carried out the following procedure. A series of oligonucleotide primers, annealed at different positions along the lacZ region of bacteriophage M13mp9 DNA, were elongated in the presence of 3 of the 4 deoxynucleoside 5′-triphosphates (dNTPs)_until one or a few misincorporations occured in each elongated primer. The elongated primers (containing deoxynucleotide residues that had been misincorporated in the presence of either Mg²⁺ or Mn²⁺) were then isolated and sequenced by the "dideoxy" chain termination method to determine the identity of deoxynucleoside monophosphates (dNMPs) that had been misincorporated at different template positions during the original 'minus' reactions, activated by Mg²⁺ or Mn²⁺. The results obtained by this approach revealed that both the type of misincorporation and the effect of substituting Mn²⁺ for Mg²⁺ depended on the nucleotide sequence of the template. At 40% of the template positions at which misincorporation was compared with both metal ions (8 out of 20), the identity of mispairs differed significantly for synthesis activated by Mn²⁺ versus Mg²⁺. Of these 8 sites, 4 exhibited increased transversions in the presence of Mn²⁺, while 4 exhibited decreased transversions with Mn²⁺.