ROLE OF DNA-REPAIR BY (A)BC EXCINUCLEASE AND OGT ALKYLTRANSFERASE IN THE FINAL DISTRIBUTION OF LACI(-D) MUTATIONS INDUCED BY N-BUTYL-N-NITROSOUREA IN ESCHERICHIA-COLI

In the absence of nucleotide excision repair, the additional deficienc y of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increase in mutation inductio n by N-butyl-N-nitrosourea (BNU), Irrespective of the presence or abse nce of the Ogt ATase, little mutagenic response was detected in Uvr(+) bacteria in the concentration range 0-8 mM BNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficient ly recognized and repaired by the nucleotide excision repair system. I ncreased susceptibility to mutagenesis by BNU was detected in Uvr(-) O gt(+) bacteria, but the Uvr(-) Ogt(-) double mutant exhibited much hig her sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM BNU within the initial part of the lad gene of E.coli were recovered from Uvr(+) Ogt(-), Uvr(-) Ogt(+) and Uvr Ogt (-) bacteria. A total of 454 independent mutations were characterized by DNA sequence analysis. The BNU-induced spectra were dominated by G: C-->A:T transitions, consistent with the major role of the O-6-alkylgu anine miscoding lesion in mutagenesis by alkylating agents. Specific s ites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically sign ificant differences among the spectra (P < 10(-6)). We examined the in fluence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-fl anking base associated with the BNU-induced G:C-->A:T transitions; pre ferences different from those previously reported for other alkylnitro soureas were detected. We discuss how these differences might be cause d by BNU producing branched chain derivatives, in addition to the expe cted linear chain adducts, and by possible preferences with respect to both the initial distribution of O-6-butylguanine lesions and their r epairability.