A forward mutation assay in phage T4: application to gene 42 mutator mutations

A forward mutation assay was developed to study mutagenic specificity induced by temperature-sensitive alleles of bacteriophage T4 gene 42, which encodes a thermolabile deoxycytidylate hydroxymethylase. Thymidine kinase (tk) mutations induced by T4 ts B3 at a semi-permissive temperature (34 degrees C) were selected under near-ultraviolet light on synthetic agar plates containing bromodeoxyuridine, and sequenced after PCR amplification of the tk gene. 21 of 23 tk- mutations identified were C-->T transitions, while the remainder were C-->A transversions. Analyses of the DNA sequence around each mutant site suggest that the mispairing of thymine with guanine in the template is suppressed when the next nucleotide is dGTP. The 5' neighbor nucleotide of the mismatch may influence mutation frequency as well; no mutations with dAMP residues on the upstream side were seen. Our observations with the forward mutation assay here are consistent with previous results from an rII reversion assay, supporting our model that the mutator phenotype displayed by tsLB3 is a consequence of perturbation of dNTP supplies to replication sites due to partial impairment of thermolabile deoxycytidylate hydroxymethylase at a semi-permissive temperature. The forward mutation assay described here is readily adapted for other studies of mutagenesis in T4 phage.