On the Role of Deoxyribonucleic Acid Polymerase in Determining Mutation Rates: CHARACTERIZATION OF THE DEFECT IN THE T4 DEOXYRIBONUCLEIC ACID POLYMERASE CAUSED BY THE TS L88 MUTATION

Abstract

Abstract The T4 bacteriophage DNA polymerase mutant ts L88 has an unusually high mutation rate and is temperature-sensitive for DNA synthesis. The ts L88 DNA polymerase has been purified to homogeneity and has been compared to the wild type T4 DNA polymerase with respect to in vitro polymerase and 3' to 5' exonuclease activity, and also with respect to frequency of utilizing deoxynucleoside triphosphates that are not complementary to homopolymer templates. For the latter, the assay used measures not only stable misincorporation, but also detects any of the noncomplementary nucleotide incorporated and subsequently hydrolyzed by the polymerase-associated exonuclease, resulting in the formation of deoxynucleoside monophosphate derived from the deoxynucleoside triphosphate substrate. A specific defect was found in the ability of the ts L88 enzyme to discriminate against utilization of dGTP and dCTP when poly(dA)·poly(dT) was used as template. These substrates were utilized at 6 times the rate of the wild type enzyme by an amount of the mutant enzyme that utilized the complementary substrates dATP and dTTP at nearly the same rate as the wild type. The two enzymes were equally efficient in excising the noncomplementary residues incorporated during the reaction. On the other hand, when a template was used to which the four substrates were complementary, no difference in recognition of specific bases by the two enzymes was detectable. Thus, with T7 DNA as template both enzymes incorporated dTMP, dGMP, and dCMP at about the same rate as dAMP. During this reaction both enzymes hydrolyzed newly added dAMP residues 2 to 4 times as frequently as dTMP and dGMP, and more than 10 times as often as newly incorporated dCMP residues. Another difference in the ability of the two enzymes to discriminate against noncomplementary, but not complementary, substrates was seen when the rates of utilization of dGTP and dATP with poly(dA)·poly(dT) by the two enzymes were compared as a function of temperature. There was a marked increase in utilization of the noncomplementary substrate between 25 and 30° by ts L88 but not by the wild type enzyme. Neither enzyme showed such a stimulation over this temperature range with the complementary nucleotide. The polymerase activity of the ts L88 enzyme was much more unstable when assayed at high temperature than was its exonuclease activity. It is concluded that both the temperature-sensitive and mutator characteristics of the ts L88 mutation result from a greater defect in polymerase than exonuclease activity.