Gw. Yang et al., Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation, BIOCHEM, 38(25), 1999, pp. 8094-8101
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.