| Summary: | Eukaryotic genomic DNA is packaged into chromatin and all aspects of DNA metabolism require chromatin modification to facilitate these processes, including DNA repair. In response to ultra violet (UV) irradiation of cells, the chromatin undergoes structural changes allowing specific factors to gain access to the DNA damage sites to enable the repair of the DNA lesions efficiently. Following repair, these events must be reversed to restore chromatin to its normal undamaged state. The UV light-induced increase in histone H3 acetylation and the subsequent chromatin alteration that is associated with it is important for efficient DNA repair by the nucleotide excision repair (NER) pathway. In wild type cells, the elevated levels of damage-induced histone H3 acetylation is restored to the pre-damage levels following repair of the damage. However, in the absence of effective repair, histone H3 acetylation levels remain constitutively high, and subsequently the chromatin structure stays in an open configuration and does not get restored to its normal undamaged state. In this study, I undertook experiments to determine the mechanism by which the structure of chromatin is restored after the repair of DNA damage in yeast cells exposed to UV-induced DNA damage. The Rad7/Rad16 containing global genome nucleotide excision repair (GG-NER) complex controls the occupancy of the histone acetyl transferase (HAT) Gcn5 on chromatin, which mediates the level of histone H3 acetylation. Moreover, the activity of histone deacetylaces (HDACs) and HATs are present in an equilibrium status, and any changes in one of them means that the balance of histone acetylation in the cell changes. Here, Chromatin Immunoprecipitation (ChIP) and ChIP-on-chip methods were adapted to study how the above factors bind to chromatin, both before and following UV irradiation in wild-type and specific DNA repair defective cells. My results show that the Rad7/Rad16-containing GG-NER complex, and Gcn5, remain bound to the chromatin in UV irradiated NER defective cells, and as a result, this keeps histone H3 acetylation status continuously high. My results also showed that, immediately following UV irradiation when histone H3 acetylation levels are increased, the histone deacetylases (Rpd3 and Hda1) levels decrease. Importantly, in wild type cells, where repair has taken place, restoration of histone H3 acetylation levels and HDAC occupancy to pre-damaged levels was observed. I showed that these changes are dependent upon active DNA repair. In conclusion, my results provide evidence that the GG-NER complex and HDACs are contributing to the regulation of UV-induced histone H3 acetylation levels in order to v control the chromatin structure following DNA damage. This suggests that these factors have a role in the access and restore phases of the ARR model.
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