Preincision Processing of Cyclobutane Pyrimidine Dimers in Escherichia coli

Abstract

AbstractIn both prokaryotes and eukaryotes cyclobutane pyrimidine dimers (CPD) formed by UV-irradiation are repaired mainly by the nucleotide excision repair (NER) pathway. Evidence has been presented that CPDs are repaired more rapidly and efficiently in vivo than when using purified NER components in vitro, and raises the possibility that CPD can be processed at the preincision stage before being removed by NER pathway. We investigated this hypothesis in E. coli cells and found that the phosphodiester bonds in CPDs are indeed incised (for the sake of simplicity we term this result as CPD*) in a time-dependent fashion in NER-proficient cells. This CPD preincision processing also occurs in NER-deficient uvrB- and uvrC-, but not in uvrA-mutant cells, and in endonuclease V (nfi) mutant cells. Introduction of the nfi mutation into wild type or uvrB-mutant cells results in disablement of CPD to CPD* conversion and enhances UV sensitization to the level of uvrA-mutant cells. We found that in a reconstituted in vitro system, that purified UvrA and Nfi are necessary but not sufficient to convert CPD to CPD*. A UvrC homologous protein, Cho, is also needed for this conversion. In addition, we found that Nfi does not bind to UV-irradiated DNA, suggesting that UvrA and Cho proteins may function as matchmakers for Nfi-CPD binding, which consequently results in the conversion of CPD to CPD*. We further demonstrated that CPD*, but not CPD, can allow efficient translesion bypass synthesis with accuracy. We thus have elucidated a new CPD preincision processing mechanism and genes involved in this process. The biological significance of these findings is discussed elsewhere (another manuscript in preparation).