INFLUENCE OF NUCLEOTIDE EXCISION-REPAIR OF ESCHERICHIA-COLI ON RADIATION-INDUCED MUTAGENESIS OF DOUBLE-STRANDED M13 DNA

To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double- stranded M13mp10 DNA, which contains a part of the Inc operon, includi ng the promoter/operator region, the lacZ alpha gene and a 144 basepai r (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N-2. Subsequently, the r adiation-exposed DNA was transfected to wild-type or NER-deficient (uv rA(-)) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutati ons obtained from wild-type and uvrA(-) E. coli appeared to consist ma inly of bp substitutions. However, in contrast to wild-type cells, a r elatively large proportion of the mutations obtained from the NER-defi cient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause thi s particular type of frameshift. Comparison of the bp substitutions be tween both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A --> C/ G transitions which are virtually absent in wild-type E. coli. This in dicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesion s which cause C/G --> T/A transitions, which make up 52% of the bp sub stitutions in uvrA(-) cells versus 17% in wild-type cells. Strikingly, C/G --> G/C transversions appeared to be only formed in wild-type, wh ere they make up 22% of all bp substitutions, and not in the NER-defic ient E. coli. This result suggests, that due to the action of NER, a p articular type of mutation may be introduced. A similar indication hol ds for C/G --> A/T transversions, which are predominant in wild-type ( 58%) and in the minority in uvrA(-) cells (15%).