Revealing the secrets of DNA repair
DNA is the major constituent of the chromosomes within the cell nucleus and plays a central role in the determination of hereditary characteristics. DNA is strongly affected by mutagens in many ways, including changes in the DNA structure of genes and interference with the coding system or causing chromosomal damage. These DNA-damaging agents may be chemicals or types of radiation such as UV-radiation. In lesions, such as in Cockayne syndrome (CS), cells are very sensitive to mutagens and are unable to restore damage by inhibiting RNA synthesis. Under the prism of repair kinetics, after induction of DNA damage, the defective initiation of the transcription mechanism was assumed to be responsible for the impaired transcription in CS cells. In other words, it was believed that, damaged DNA delayed transcription initiation and caused cell repair failure. Within the framework of this project researchers tested this hypothesis by studying the effect of a UV irradiation on transcription. By employing an in vitro transcription system they managed to uncouple the initiation of transcription from later effects. Since transcription kinetics in nuclear extracts imitate those in intact cells, they investigated nuclear extracts coming from UV-irradiation for both normal human and CS cells in order to assess transcription activity. The results showed that in normal cells 1 hour after UV exposure, a decreased transcription activity was present and 6 hours after exposure transcription activity was completely restored. On the other hand, at any time after UV exposure extracts from CS (impaired) cells reduced transcription activity was observed. Moreover, the reduced transcription activity was accompanied by a strong reduction of a form of RNA polymerase II, that is used in the initiation complex. These findings prove that UV-induced inhibition of transcription involved both repression of transcription initiation and the effect of phosphorylation of RNA polymerase II in the transcription initiation activity. This means that blocking the transcription initiation is one of the factors that inhibit the repair process. Moreover, production of hypophosphorylated RNA polymerase II after DNA damage, seems to be the most essential reason for restoring the initiation of the transcription activity. Whether CS proteins are required for this process is a yet unknown. The research work performed also used a novel technique of local UV-irradiation combined with fluorescent antibody labelling for studying the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells. Their findings identified the most important NER factor in the reaction mechanism for the recruitment of all subsequent NER factors including transcription-repair factors. Finally, these investigations gave a better insight in the hypothesis of sequential assembly of repair proteins at the site of the damage. The project results have a wide range of applications in the fields of biotechnology, particularly for genetics and protein engineering. Better understanding of the micro-world of cells can contribute significantly in the design and implementation of new therapies for untreated diseases, such as cancer.
