Table 1.

Fidelities of purified pol β-WT and pol β-14 in vitro

Type of error	Assay	Error frequency, ×10−4		Increase, -fold
		pol β-WT	pol β-14
Base substitution	TGA reversion	1.9 ± 0.10	87 ± 19	46
	G:N kinetics	9.8 ± 6.0	830 ± 460	85
Frameshift	TTTTT reversion	1.9 ± 0.7	470 ± 20	240
All	tk loss of function	21 ± 0.5	540 ± 0.7	25

The TGA target is in a modified version of the lacZα gene and refers to the opal codon reversion assay which detects base substitution mutations. The TTTTT target is in a modified version of the lacZα gene and refers to the frameshift reversion assay which detects predominantly frameshift mutations. For M13mp2-derived assays with pol β-WT, at least 85,000 plaques were scored; with pol β-14, at least 15,000 plaques were scored. At least 30,000 colonies were analyzed for each HSV-tk experiment. Data represent the averages of two independent experiments and are presented with standard errors. The values for the kinetic assay are the average nucleotide misinsertion frequencies (f) of the three G:N mispairs examined. Initial evaluation of the polymerase activities of these recombinant proteins on activated calf thymus DNA showed that the K_{m (dTTP)} for pol β-14, 1.9 ± 0.3 μM, is quite similar to that of pol β-WT (1.2 ± 0.2 μM). In contrast, the mutant protein exhibited a K_{m (activated DNA)} of 80 ± 6.5 μM and k_cat of 0.0015 sec⁻¹, which are significantly lower than those of the pol β-WT protein (362 ± 15 μM and 0.03 sec⁻¹, respectively). Our recombinant pol β-WT protein and an independent preparation lacking the extra amino acids at the amino terminus (supplied by Akio Matsukage, Aichi Cancer Center Research Institute, Nagoya, Japan) yielded essentially identical values in these kinetic assays (data not shown). Initial evaluation of polymerase activities on the DNA substrate employed in the kinetic fidelity assay showed similar trends as that observed with activated DNA. The K_{m (app)} was 190 ± 45 μM for pol β-WT and 83 ± 27 μM for pol β-14. The k_cat for WT using this DNA substrate was 2.6 sec⁻¹, and for pol β-14 in the k_cat was 0.055 sec⁻¹.