Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation

The function of six highly conserved residues (Arg482, Lys483, Lys486, Lys5 60, Asn564, and Tyr567) in the fingers domain of bacteriophage RB69 DNA pol ymerase (RB69 gp43) were analyzed by kinetic studies with mutants in which each of these residues was replaced with Ala. Our results suggest that Arg4 82, Lys486, Lys560, and Asn564 contact, the incoming dNTP during the nucleo tidyl transfer reaction as judged by variations in apparent K-m and k(cat) values for dNTP incorporation by these mutants compared to those for the ex onuclease deficient parental polymerase under steady-state conditions. On t he basis of our studies, as well as on the basis of the crystal structure o f RB69 gp43, we propose that a conformational change in the fingers domain, which presumably occurs prior to polymerization, brings the side chains of Arg482, Lys486, Lys560, and Asn564 into the vicinity of the primer-templat e terminus where they can contact the triphosphate moiety of the incoming d NTP. in particular, on the basis of structural studies reported for the "cl osed" forms of two other DNA polymerases and from the kinetic studies repor ted here, we suggest that (i) Lys560 and Asn564 contact the nonbonding oxyg ens of the alpha and beta phosphates, respectively, and (ii) both Arg482 an d Lys486 contact the gamma phosphate oxygens of the incoming dNTP of RB69 g p43 prior to the nucleotidyl transfer reaction. We also found that Ala subs titutions at each of these four RB69 gp43 sites could incorporate dGDP as a substrate, although with markedly reduced efficiency compared to that with dGTP, In contrast in the parental exo(-) background, the K483A and Y567A s ubstituted enzymes could not use dGDP as a substrate fur primer extension. These results, taken together, are consistent with the putative roles of th e four conserved residues in RB69 gp43 as stated above.