No abstract available

[en] The induction and removal of UV-induced photolesions was investigated in confluent human fibroblasts employing two different approaches. Photolyase and highly purified E. coli Uvr A,B and C proteins were used to measure the frequency of 6-4 photoproducts (6-4PP) in transcriptionally active and inactive genes. At a UV-dose range of 20-60 J/m² 6-4PP were induced at about 4-fold lower frequency then cyclobutane pyrimidine dimers (CPD). In normal cells exposed to 30J/m² 6-4PP were induced at about 4-fold lower frequency then cyclobutane pyrimidine dimers (CPD). In normal cells exposed to 30J/m², the repair of 6-4PP was very rapid in both active and inactive genes when compared to CPD removal. No strand specific repair of 6-4PP in active genes was observed, although repair of 6-4PP occurred preferentially in the active genes when compared to inactive X-chromosomal genes. In xeroderma pigmentosum group C cells 6-4PP were selectively removed from the transcribed strand of active genes. In these cells the kinetics of repair of CPD and 6-4PP from the transcribed strand of active genes was very similar. Our results indicate that 6-4PP can be removed by a transcription coupled repair pathway, but that repair of 6-4PP by the global repair system is much more efficient. The sam conclusion can be drawn from studies aimed to determine BUdR labelled repair sites in genomic fragments. The results of these studies indicate a lack of strandspecific repair of 6-4 PP in active genes in normal cells at a relative low UV-dose of 10J/m²

[en] The pathway of nucleotide excision repair (NER) consists of two sub pathways i.e. global genome repair (GGR) and transcription coupled repair (TCR). GGR removes DNA damage from the genome overall while TCR is responsible for the accelerated removal of lesions from the transcribed strand of expressed genes. We have analysed the assembly of the GGR complex in normal and DNA repair-deficient (xeroderma pigmentosum) human cells, employing a technique of local UV-irradiation and immuno staining. Local exposure of small parts of the nucleus of normal human cells causes rapid accumulation of NER factors to sites of local damage. This accumulation disappears after about 2 to 3 hours, a time frame which is similar to that observed for the repair of 6-4 photo products. Comparison of cells from various xeroderma pigmentosum complementation groups showed that XPC is essential for NER complex formation at sites of local damage. Furthermore, DDB2 which is deficient in XP-E cells, binds to DNA damage before XPC and facilitates NER complex formation. Recognition of UV-induced DNA damage during TCR does not need the presence of XPC and is probably mediated by RNA polymerase II (RNAPII) stalled at the site of a lesion. Upon removal of the lesion transcription is resumed. This is in line with the observation that Cockayne syndrome cells are unable to carry out TCR and are also defective in restart of transcription following inhibition after UV-exposure. Since it has been shown that TCR is also observed after introduction of base damage by exposure to ionising radiation, we investigated the frequency of base damage as a function of dose following X-rays. Subsequently, we determined the inhibition of RNA synthesis at a dose of X-rays causing equal frequency of base damage as found after exposure to 10 J/m2 UV-radiation. The results show that inhibition of transcription following exposure to X-rays is very limited and much less than observed after exposure to UV-radiation when compared at equal levels of DNA base damage. Furthermore, CS cells behave in this respect in the same way as normal cells or cells defective in Ligase 4. Analysis of cell free extracts showed that the unphosphorylated form of RNAPII disappears after UV-radiation but not following exposure to X-rays. These data suggest that TCR of base damage induced by X-rays is not dependent on stalling of RNAPII at the site of a lesion

[en] The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimer-specific endonuclease V of bacteriophage T₄. The results suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts the authors observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in regions of the genome. (Auth.)

No abstract available

[en] Ionizing radiations induced a spectrum of lesions in the DNA and among these DNA double strand breaks (DSBs) appear to be the most important lesion leading to chromosomal aberrations. Several experimental evidences which support this contention will be presented. In human peripheral blood lymphocytes, it is seen that the kinetics of repair of DNA strand breaks (as measured by nucleoid sedimentation) parallels the repair of chromosome breaks (as measured by metaphase analysis as well as by premature chromosome condensation (PCC) techniques). Introduction of restriction endonucleases (which introduce only DNA DSBs) into permeabilized CHO cells leads to chromosomal aberrations. The pattern of production of these aberration, i.e., chromosome type G1 and chromatid type in G2 is similar to that induced by ionizing radiations. X-rays sensitive CHO mutants which are deficient in repair of DSBs respond with increased frequency of X-ray induced aberrations in comparison to the wild type cells. We have studied the kinetics of formation of dicentrics and fragments in human lymphocytes using PCC technuque. Following irradiation with 2 Gy most of the dicentrics are formed immediately whereas the fragments are repaired slowly up to 2 hrs. (authors)

[en] The effects of sodium butyrate-mediated alterations in chromatin structure on the yields of X-ray-induced chromosomal abberrations were studied in human peripheral blood lymphocytes. The results show that sodium butyrate pre-treatment leads to a significant increase in the frequencies of dicentrics and rings, but not of fragments. The data from biochemical studies suggest that the numbers and rates of repair of X-ray-induced DNA-strand breaks are the same in butyrate-treated and untreated cells. The authors suggest that the observed effect is probably a consequence of butyrate-induced conformational changes in the chromatin of G₀ lymphocytes. (Auth.)

[en] The effect of caffeine on radiation-induced chromosomal aberrations and DNA strand breaks in unstimulated human lymphocytes was investigated. When present prior to and during the radiation exposure, caffeine treatment was found to cause either potentiation or protection against induction of chromosomal aberrations depending on the concentration and temperature. When the nucleoid sedimentation technique was applied, enhancement or reduction of radiation-induced DNA strand breaks by caffeine was also found to be dependent on temperature and caffeine concentration. It is proposed that caffeine, in addition to its suspected ability to influence DNA repair, can also influence the induction of DNA damage, leading to alterations in the yield of chromosomal aberrations

[en] Short treatment of cytosine arabinoside (araC) increases the frequencies of aberrations induced by X-rays in human lymphocytes, evaluated at the first mitosis following stimulation, or as prematurely condensed chromosomes of G₀ nuclei. Parallel biochemical experiments using nucleoid sedimentation technique, demonstrate that araC inhibits rejoining of DNA-strand breaks effectively. These results point out that X-ray-induced short-lived DNA strand breaks lead to chromosomal aberrations in human lymphocytes. (Auth.)

[en] Sister chromatid exchanges (SCEs) are cytological manifestations of DNA double-strand breakage and rejoining at homologous sites between the two chromatids of a chromosome. The occurrence of SCEs was deduced from the transformation of small ring chromosomes to large ring chromosomes following cell division. Using tritiated thymidine as marker and microautoradiography for detection, others demonstrated the occurrence of SCEs from the silver grain pattern on the sister chromatids. This method was eventually replaced by cytochemical methods. One showed that if cells were grown in medium containing 5-bromo-deoxyuridine (BrdUrd) for two cycles, the sister chromatids can be distinguished by the differential quenching of the fluorescence of the fluorochrome Hoechst 33258. Reduced staining with Giemsa stain of the BrdUrd-incorporated chromatids was also found to be useful in differentiating sister chromatids. The authors review in this chapter only those studies which have implications on the origin of SCEs