The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZ alpha gene

Base excision repair (BER) is a very important repair mechanism to remove o xidative DNA damage. A major oxidative DNA damage after exposure to ionizin g radiation is 7,8-dihydro-8-oxoguanine (8oxoG). 8oxoG is a strong mutageni c lesion, which may cause G:C to T:A transversions if not repaired correctl y. Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG. In the past years, e vidence evolved that nucleotide excision repair (NER), a repair system orig inally thought to repair only bulky DNA lesions, can also repair some oxida tive DNA damages. Examples of DNA damages which are recognized by NER are t hymine glycol and abasic sites (AP sites). The main objective of this study is to determine if NER can act as a backup system for the repair of sponta neous and gamma-radiation-induced damages when Fpg is deficient. For that p urpose, the effect of a NER-deficiency on the spontaneous and gamma-radiati on-induced mutation spectrum in the lacZ gene was determined, using double- stranded (ds) M13 DNA, with the lacZ alpha gene inserted as mutational targ et sequence. Subsequently the DNA was transfected into a fpg(-)uvrA(-) Esch erichia coli strain (BH420) and the mutational spectra were compared with t he spectra of a fpg(-) E. coli strain (BH410) and a wild type E, coli strai n (JM105), which were determined in an earlier study. Furthermore, to exami ne effects which are caused by UvrA-deficiency, and not by Fpg-deficiency, the spontaneous and gamma-radiation-induced mutation spectra of an E. coli strain in which only UvrA is deficient (BH430) were also determined and com pared with a wild type E. coli strain (JM105). The results of this study in dicate that if only UvrA is deficient, there is an increase in spontaneous G:C to T:A transversions as compared to JM105 and a decrease in A:T to G:C transitions. The gamma-radiation-induced mutation spectrum of BH420 (fpg(-) uvrA(-)) shows a significant decrease in G:C to A:T and G:C to T:A mutation s, as compared to BH410 where only Fpg is deficient. Based on these results , we conclude that in our experiments NER is not acting as a backup system if Fpg is deficient. Instead, NER seems to make mistakes, leading to the fo rmation of mutations. (C) 2000 Elsevier Science B.V. All rights reserved.