title: Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I.
abstract: Incorporation of dideoxynucleotides by T7 DNA polymerase and Escherichia coli DNA polymerase I is more efficient when Mn2+ rather than Mg2+ is used for catalysis. Substituting Mn2+ for Mg2+ reduces the discrimination against dideoxynucleotides approximately 100-fold for DNA polymerase I and 4-fold for T7 DNA polymerase. With T7 DNA polymerase and Mn2+, dideoxynucleotides and deoxynucleotides are incorporated at virtually the same rate. Mn2+ also reduces the discrimination against other analogs with modifications in the furanose moiety, the base, and the phosphate linkage. A metal buffer, isocitrate, expands the MnCl2 concentration range effective in catalyzing DNA synthesis. The lack of discrimination against dideoxynucleoside triphosphates using T7 DNA polymerase and Mn2+ results in uniform terminations of DNA sequencing reactions, with the intensity of adjacent bands on polyacrylamide gels varying in most instances by less than 10%.
authors.affiliation: Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115.
authors.forename: S
authors.initials: S
authors.lastname: Tabor
authors.forename: C C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D004256:DNA Polymerase I / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology*; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*
chemical_list: D003854:Deoxyribonucleotides; D008345:Manganese; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium