Fidelity studies of the human DNA polymerase alpha. The most conserved region among alpha-like DNA polymerases is responsible for metal-induced infidelity in DNA synthesis

W.C Copeland,N.K Lam,T.S Wang

doi:10.1016/s0021-9258(18)82089-0

Abstract

Mutational studies in the highly conserved region I domain of the human DNA polymerase alpha enzyme demonstrated a change in metal cation-specific catalysis. Here, we extend the investigation to include the fidelity of DNA synthesis by these mutants, studying misinsertion, mispair extension, and the nucleotide analog utilization. The fidelity of region I mutants and wild type human DNA polymerase alpha enzyme were analyzed with either Mg2+ or Mn2+ as the metal activator. Despite the known mutagenic effect of Mn2+ in causing polymerases to misinsert nucleotides and to utilize dideoxynucleotides, we have found that two region I mutants, D1002N and T1003S, which utilize Mn2+ in catalysis more effectively than Mg2+, actually have a 70- and 40-fold higher misinsertion fidelity, respectively, in Mn(2+)-catalyzed reactions than that of the wild type enzyme. The enhanced misinsertion fidelity of these two mutants in Mn(2+)-catalyzed reactions is due to Km discrimination of the incorrect nucleotide where the D1002N and T1003S had a 850- and 62-fold higher Km for insertion of incorrect than correct nucleotide, respectively. In Mg(2+)-catalyzed reactions, all of the region I mutants exhibited similar misinsertion efficiencies as the wild type polymerase. Study of mispair extension showed that in Mn(2+)-catalyzed ractions, the wild type polymerase alpha enzyme readily extended mispair termini. In contrast, the two region I mutants, D1002N and T1003S, were unable to extend the mispaired termini in either Mg(2+)- or Mn(2+)-catalyzed reactions. These results suggest that the side chains of region I amino acids play an essential role in the Mn(2+)-induced infidelity during DNA synthesis by human DNA polymerase alpha. The effects of the metal activator on the utilization of two nucleotide analogs, 3'-azido-3'-deoxythymidine triphosphate and ddCTP, by the region I mutants were also investigated.

Highlights

Fidelity Studiesof the Human DNA Polymerase cy THE MOST CONSERVED REGION AMONG a-LIKE DNA POLYMERASES IS RESPONSIBLE FOR METAL-INDUCEDINFIDELITY IN DNA SYNTHESIS*
We extend the investigation toinclude the fidelity of DNA synthesis by these mutants, studyingmisinsertion, mispair extension, and thenucleotide analog utilization
Despite the known mutagenic effect of Mn2+in causing polymerases to misinsert nucleotides and toutilize dideoxynucleotides, we have found that two region I mutants, D1002N and T1003S, which utilize Mn2+in catalysis more effectively than Mg2+, have a 70- and 40-fold higher misinsertion fidelity, respectively, in Mn2+-catalyzedreactions than that of the wild type enzyme

Summary

Introduction

Fidelity Studiesof the Human DNA Polymerase cy THE MOST CONSERVED REGION AMONG a-LIKE DNA POLYMERASES IS RESPONSIBLE FOR METAL-INDUCEDINFIDELITY IN DNA SYNTHESIS*. In Mc-catalyzedreactions with running start primer-template, the misinsertion efficiencies of the incorrect dCMPby wild type polymerase cy and the four mutants were in the same order of magnitude (Table 1).

Results

Conclusion