Anneke van Hoffen

(6-4) Photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.

Differential Repair of the Two Major UV-Induced Photolesions in Trichothiodystrophy Fibroblasts

Defects in nucleotide excision repair have been shown to be associated with the photosensitive form of the disorder trichothiodystrophy (TTD). Most repair-deficient TTD patients are mutated in the XPD gene, a subunit of the transcription factor TFIIH. Knowledge of the kinetics and efficiency of repair of the two major UV-induced photolesions in TTD is critical to understand the role of unrepaired lesions in the process of carcinogenesis and explain the absence of enhanced skin cancer incidence in TTD patients contrarily to the xeroderma pigmentosum D patients. In this study, we used different approaches to quantify repair of UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6–4PP) at the gene and the genome overall level. In cells of two TTD patients, repair of CPD and 6–4PP was reduced compared with normal human cells, but the reduction was more severe in confluent cells than in exponentially growing cells. Moreover, the impairment of repair was more drastic for CPD than 6–4PP. Most notably, exponentially growing TTD cells displayed complete repair 6–4PP over a broad dose range, albeit at a reduced rate compared with normal cells. Strand-specific analysis of CPD repair in a transcriptional active gene revealed that TTD cells were capable to perform transcription-coupled repair. Taken together, the data suggest that efficient repair of 6–4PP in dividing TTD cells in concert with transcription-coupled repair might account for the absence of increased skin carcinogenesis in TTD patients.

Nucleotide excision repair of UV-radiation induced photolesions in human cells

Abstract Nucleotide excision repair (NER) is a versatile and highly conserved repair system capable of removing a wide range of DNA lesions that distort the stacking of the DNA double helix. These lesions include cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) which are the consequence of exposure to solar radiation. Although specific processes for repair of UV-induced photo lesions have evolved in a number of species (including photolyase-type enzymes in Escherichia coli , yeast and Drosophila ), repair of CPD and 6-4PP in human cells is completely dependent on NER. The crucial role of NER in the protection against the deleterious effects of sunlight is best demonstrated by the photosensitive hereditary human syndrome xeroderma pigmentosum (XP). XP-patients are defective in NER and exhibit a strong predisposition to skin cancer by sunlight. Two NER subpathways have been identified, i.e. global genome repair which removes lesions from all parts of the genome and transcription coupled repair which causes accelerated repair of lesions from the transcribed strand of expressed genes. This chapter provides a description of the molecular mechanism of NER in mammalian cells including the major proteins involved and their functioning in the various steps of NER.