DNA-REPAIR BY OGT ALKYLTRANSFERASE INFLUENCES EMS MUTATIONAL SPECIFICITY

Forward mutations induced by ethylmethane sulfonate (EMS) in the lad g ene of Escherichia coli were recovered from bacteria proficient or def icient in the alkyltransferase encoded by the constitutive ogt gene, E MS doses of 100 or 200 mM (Ogt(+)) and of 50 mM (Ogt(-)) were selected from the corresponding dose-response curves for DNA sequence analysis , A total of 239 induced mutations affecting the N-terminal region of the lad gene were characterized, All mutations were G:C-->A:T transiti ons, consistent with the predominant role of the O-6-ethylguanine misc oding lesion in mutagenesis by EMS. In the Ogt(+) spectrum at the lowe st tested dose of 100 mM EMS, guanines preceded by an A or T base at t he 5' side were on average 3.2 times more likely to mutate than those preceded by a G or C base, This bias diminished at the higher EMS dose (200 mM) and disappeared in the Ogt(-) genetic background, Previously reported data for Ogt(+) bacteria in a Uvr-proficient background show an opposite bias in favor of mutations at guanines preceded by a G or C base, The overall 5' flanking base influence was estimated as 8-fol d. These data suggest that DNA repair by Ogt alkyltransferase plays an important role in the processing of ethylation-induced lesions respon sible for GC-->AT transitions, influencing their ultimate distribution with respect to sequence context, The data further suggest that Ogt a nd UvrABC excision repair, the two major mechanisms of protection agai nst the biological consequences of long-chain alkylating agents, show different DNA sequence specificity and that the relative importance of these two systems is highly dependent upon the chemical dose.