Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities.