Abstract
The N-(deoxyguanosine-8-yl)-2-acetylaminofluorene (dG-C8-AAF) lesion is among the most helix distorting DNA lesions. In normal fibroblasts dG-C8-AAF is repaired rapidly in transcriptionally active genes, but without strand specificity, indicating that repair of dG-C8-AAF by global genome repair (GGR) overrules transcription-coupled repair (TCR). Yet, dG-C8-AAF is a very potent inhibitor of transcription. The target size of inhibition (45 kilobases) suggests that transcription inhibition by dG-C8-AAF is caused by blockage of initiation rather than elongation. Cockayne's syndrome (CS) cells appear to be extremely sensitive to the cytotoxic effects of dG-C8-AAF and are unable to recover inhibited RNA synthesis. However, CS cells exhibit no detectable defect in repair of dG-C8-AAF in active genes, indicating that impaired TCR is not the cause of the enhanced sensitivity of CS cells. These and data reported previously suggest that the degree of DNA helix distortion determines the rate of GGR as well as the extent of inhibition of transcription initiation. An interchange of the transcription/repair factor TFIIH from promoter sites to sites of damage might underlie inhibition of transcription initiation. This process is likely to occur more rapidly and efficiently in the case of strongly DNA helix distorting lesions, resulting in a very efficient GGR, a poor contribution of TCR to repair of lesions in active genes, and an efficient inhibition of transcription.
Highlights
Nucleotide excision repair (NER)1 constitutes a versatile process capable of recognizing and eliminating a broad spec
We show that repair of a major fraction of dG-C8-AAF lesions is very efficient and occurs without strand specificity, probably because the transcription-coupled repair (TCR) is overruled by global genome repair (GGR)
Formation of DNA Adducts—Because incubation of cells with NA-AAF leads primarily to the induction of dG-C8-AF, we had to develop a system of inducing dG-C8-AAF
Summary
Nucleotide excision repair (NER) constitutes a versatile process capable of recognizing and eliminating a broad spec-. Understanding the mechanisms and identification of the factors that determine DNA damage recognition requires lesionspecific information on repair kinetics, transcription blockage capacity, and structural distortion of the DNA helix. Exposure of cells to NA-AAF provides the possibility of studying structurally different DNA lesions, for which detailed information exists on DNA conformation and transcription/replication blockage (for review, see Ref. 5). Formation of dG-C8-AAF lesions leads to a conformation change of the DNA which results in a local denaturation of about 5 base pairs [6, 7]. This denaturation is caused by stacking of the aminofluorene group inside the DNA-helix, a conformation that is stabilized by the acetyl group. In rat the high frequency of formation of dG-C8-AAF is caused by the presence of high levels of sulfotransferase activity, which prevents the deacetylation process by forming a stable sulfonated and acetylated compound [5]
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