ENZYME-DNA INTERACTIONS REQUIRED FOR EFFICIENT NUCLEOTIDE INCORPORATION AND DISCRIMINATION IN HUMAN DNA-POLYMERASE-BETA

In the crystal structure of a substrate complex, the side chains of re sidues Asn(279), Tyr(271), and Arg(283) of DNA polymerase beta are wit hin hydrogen bonding distance to the bases of the incoming deoxynucleo side 5'-triphosphate (dNTP), the terminal primer nucleotide, and the t emplating nucleotide, respectively (Pelletier, H., Sawaya, M., R., Kum ar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903), W e have altered these side chains through individual site-directed muta genesis, Each mutant protein was expressed in Escherichia coil and was soluble, The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis, A reversion ass ay was developed on a short (5 nucleotide) gapped DNA substrate contai ning an opal codon to assess the effect of the amino acid substitution s on fidelity, Substitution of the tyrosine at position 271 with pheny lalanine or histidine did not influence catalytic efficiency (k(cat)/K -m) or fidelity, The hydrogen bonding potential between the side chain of Asn(279) and the incoming nucleotide was removed by replacing this residue with alanine or leucine, Although catalytic efficiency was re duced as much as 17-fold for these mutants, fidelity was not, In contr ast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg(283) (Ala > Leu > Lys), The fidelity and catalytic efficiency of the alanine mutant of Arg(283) decreased 160- and 5000- fold, respectively, relative to wild-type enzyme, Sequence analyses of the mutant DNA resulting from short gap filling synthesis indicated t hat the types of base substitution errors produced by the wild-type an d R283A mutant were similar and indicated misincorporations resulting in frequent T . dGTP and A . dGTP mispairing. With R283A, a dGMP was i ncorporated opposite a template thymidine as often as the correct nucl eotide. The x-ray crystallographic structure of the alanine mutant of Arg(283) verified the loss of the mutated side chain, Our results indi cate that specific interactions between DNA polymerase beta and the te mplate base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency an d nucleotide discrimination.