N-nitrosocarbaryl-induced mutagenesis in Haemophilus influenzae strains deficient in repair and recombination

Abstract

Mutagenesis was studied in repair- and recombination-deficient strains of Haemophilus influenzae after treatment with N-nitrosocarbaryl (NC). Three different strains of H. influenzae carrying mutations affecting excision-repair of UV-induced pyrimidine dimers exhibited normal repair of premutational lesions (as detected by decreased mutation yield resulting from post-treatment DNA synthesis delay) and normal nonreplicative mutation fixation. This indicates that neither of these phenomena are caused by the same repair mechanism that removes UV-induced pyrimidine dimers from the DNA. The recombination-deficient mutant rec1 is apparently deficient in the replication-dependent mode of NC-induced mutation fixation. This conclusion is based on the following results: (1) NC-induced mutagenesis is lower in the rec1 strain than in rec + cells. (2) Repair of premutational lesions (which depends on the existence of replication-dependent mutation fixation for its detection) was not detected in the rec1 strain. (3) When nonreplicative mutation fixation and final mutation frequency were measured in the same experiment, about 1 4 to 1 3 of the final mutation yield could be accounted for by nonreplicative mutation fixation in the rec + strain, whereas all of the mutation could be accounted for in the rec1 strain by the nonreplicative mutation fixation. (4) When mutation fixation in strain dna9 rec1 was followed at the permissive (36°) and nonpermissive (41°) temperatures, it became apparent that in the rec1 strain replication-dependent mutation fixation occurs at early times, but these newly fixed mutations are unstable and disappear at later times, leaving only the mutations fixed by the nonreplicative process. The rec1 strain exhibits normal repair of NC-induced single-strand breaks or alkali-labile bonds in the DNA labeled before treatment, but is slow in joining discontinuities present in DNA synthesized after treatment. The results are consistent with the idea that in NC-treated H. influenzae cells the replication-dependent mode of mutation fixation occurs by error-prone joining of interruptions present in the DNA synthesized after treatment. The possibility still exists, however, that during DNA replication mispairing occurs opposite certain alkylation-induced lesions and that mutations arising during replication of strain rec1 later disappear as a result of degradation of newly synthesized DNA, which is excessive in this strain.