Repair of Genotoxic Damage in Chromatin

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DESCRIPTION (provided by applicant): Genome of living organisms is intrinsically unstable and subjected to incessant and deleterious damage from within and outside of cells. Fortunately, elaborate DNA repair systems, including the versatile nucleotide excision repair (NER) have evolved to overcome the consequences of genomic instability- a known hallmark of human cancer. Nevertheless, genomic repair is complicated because the DNA lesions must be promptly accessed within the highly condensed chromatin of cell's nucleus. Thus, studies are proposed to extend the scope of our ongoing work on the regulation of NER by incorporating the latest developments in DNA repair and related areas, especially chromatin remodeling. The project is based on the premise that cells rely on specialized molecular apparatus to modify/remodel/rearrange chromatin structure to overcome the barrier presented by repressive chromatin structure and thus allowing the operation of all the DNA templated processes, including DNA repair. The specific hypotheses addressed are: (i) subsequent to damage induction, e.g., by ultraviolet (UV) irradiation, nucleosomes are differentially rearranged at the sites of non-damaged and damaged chromatin, (ii) the slow and fast repair rates of two main UV photolesions, cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone photoproducts (6-4PP), are dependent on different chromatin remodeling machineries, (iii) ubiquitin-proteasome pathway (UPS) intimately regulates chromatin remodeling during NER, and (iv) the excision repair outcome is determined by the interplay between DNA damage response and chromatin remodeling. The proposed work will utilize a relevant plethora of state-of-the art technologies to address following inter-related specific objectives: (1) to demonstrate the involvement of histone modifications in the process of NER in mammalian cells, (2) to discern the nucleosome rearrangement in undamaged and damaged chromatin following UV irradiation and its effect on NER factor binding, (3) to show the participation of chromatin remodeling machinery in the repair of CPD and 6-4PP, (4) to delineate the role of UPS related chromatin remodeling in NER of different UV photolesions, and (5) to understand the interplay of checkpoint signaling, chromatin remodeling and NER factor recruitment following UV irradiation. These studies will provide seminal insights into intricate cellular processes that preserve genomic integrity and prevent initiation of human cancers.