Every day, normal cellular metabolism produces 30,000 or more oxidative lesions In the DNA of each human cell. Most of these lesions are removed by the base excision repair (BER) machinery. Certain defective BER enzymes have been shown to be oncogenic, Nucleosomes that package DNA into chromafin provide little if any protection from oxidative damage. Project 4 was added to the original POI in September, 2007, to identify rate-limiting steps in the BER of lesions in nucleosomes, and to investigate mechanisms that cells use to circumvent these limits. Studies to date indicate that the human bifunctional DNA glycosylase hNTHI can excise oxidative lesions from nucleosomal DNA without irreversibly disrupting the nucleosome, and that spontaneous, transient partial unwrapping of DNA from the histone octamer facilitates access to sterically occluded lesions. With these results as a foundation, the goals for this new proposal are to test HYPOTHESIS 1, that during BER of lesions in nucleosomes, there is a stepwise hand-off of substrates from one enzyme to the next, facilitated by BER accessory factors. In the course of testing this hypothesis, we will investigate changes in nucleosome structure that accompany individual steps in BER, These structural analyses will help guide future studies of histone chaperones and chromatin remodeling agents that facilitate BER in vivo. We also will test HYPOTHESIS 2, that histone primary sequence variants associated with either transcriptionally active or inactive chromatin influence the capacity of selected human DNA glycosylases to repair oxidative damages in nucleosomes. Information from Project 2 will help elucidate the molecular basis for interactions between hNTHI and lesion-containing nucleosomes. Information and reagents provided by Core A and Projects 1 and 3 will enable us to test HYPOTHESIS 3, that DNA polymerase p (Pol P) variants with demonstrated oncogenic potential differ from wild type Pol p in BER of lesions in nucleosomes. We also will determine If, in the context of nucleosomal templates, Pol p (or oncogenic variants of Pol P) can bind or recruit hRadSI. Core B will provide critical reagents for all of the studies in Project 4.
The information generated by the proposed studies will advance our understanding of the carcinogic consequences of damages arising from endogenous metabolism and radiafion exposure. Additionally, radiation threatment is widely used in cancer therapy. The proposed studies will advance our understanding of factors that affect radiafion sensitivity of cells and modulation of such factors could help maximize therapeutic gain during radiotherapy.
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