DNA polymerase beta: Contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity

The specific catalytic roles of two groups of DNA polymerase beta active si te residues identified from crystal structures were investigated: residues possibly involved in DNA template positioning (Lys280, Asn294, and Glu295) and residues possibly involved in binding the triphosphate moiety of the in coming dNTP (Arg149, Ser180, Arg183, and Ser188). Eight site-specific mutan ts were constructed: K280A, N294A, N294Q, E295A, R149A, S180A, R183A, and S 188A. Two-dimensional NMR analysis was employed to show that the global con formation of the mutants has not been perturbed significantly. Presteady-st ate kinetic analyses with single-nucleotide gapped DNA substrates were then performed to obtain the rate of catalysis at saturating dNTP (k(pol)), the apparent dissociation constant for dNTP (K-d), catalytic efficiency k(pol) /K-d, and fidelity. Of the three template-positioning residues, Asn294 and Glu295 (but not Lys280) contribute significantly to k(pol). Taken together with other data, the results suggest that these two residues help to stabil ize the transition state during catalysis even though they interact with th e DNA template backbone rather than directly with the incoming dNTP or the opposite base on the template. Furthermore, the fidelity increases by up to 19-fold for N294Q due to differential k(pol) effects between correct and i ncorrect nucleotides. Of the four potential triphosphate-binding residues, Ser180 and Arg183 contribute significantly to k(pol) while the effects of R 149A are relatively small and are primarily on K-d, and Ser188 appears to p lay a minimal role in the catalysis by pol beta. These results identify sev eral residues important for catalysis and quantitate the contributions of e ach of those residues. The functional data are discussed in relation to the prediction on the basis of available crystal structures.