PMID:10882852
| Citation |
Kuipers, GK, Slotman, BJ, Poldervaart, HA, van Vilsteren, IM, Reitsma-Wijker, CA and Lafleur, MV (2000) The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZalpha gene. Mutat. Res. 460:117-25 |
|---|---|
| Abstract |
Base excision repair (BER) is a very important repair mechanism to remove oxidative DNA damage. A major oxidative DNA damage after exposure to ionizing radiation is 7,8-dihydro-8-oxoguanine (8oxoG). 8oxoG is a strong mutagenic lesion, which may cause G:C to T:A transversions if not repaired correctly. Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG. In the past years, evidence evolved that nucleotide excision repair (NER), a repair system originally thought to repair only bulky DNA lesions, can also repair some oxidative DNA damages. Examples of DNA damages which are recognized by NER are thymine 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 spontaneous and gamma-radiation-induced damages when Fpg is deficient. For that purpose, the effect of a NER-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene was determined, using double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target sequence. Subsequently the DNA was transfected into a fpg(-)uvrA(-) Escherichia coli strain (BH420) and the mutational spectra were compared with the spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105), which were determined in an earlier study. Furthermore, to examine 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 compared with a wild type E. coli strain (JM105). The results of this study indicate 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 mutations, 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 formation of mutations. |
| Links | |
| Keywords |
Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; DNA Damage/genetics; DNA Damage/radiation effects; DNA Mutational Analysis; DNA Repair/genetics; DNA, Bacterial/genetics; DNA, Bacterial/radiation effects; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; DNA-Formamidopyrimidine Glycosylase; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/radiation effects; Escherichia coli Proteins; Gamma Rays; Lac Operon/genetics; Lac Operon/radiation effects; Mutation/genetics; Mutation/radiation effects; N-Glycosyl Hydrolases/deficiency; N-Glycosyl Hydrolases/genetics; Reactive Oxygen Species/metabolism; Transformation, Bacterial |
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