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.
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.
Department of Pathology, Stanford University School of Medicine, California 94305.
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WC
Copeland
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NK
Lam
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Wang
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D054306:Dideoxynucleotides; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism; D015215:Zidovudine / Q000031:analogs & derivatives / Q000378:metabolism
D054306:Dideoxynucleotides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D004247:DNA; D004257:DNA Polymerase II