Dissertation
Regulation of Base Excision Repair in Translationally and Rotationally Positioned DNA Lesions in Nucleosome Core Particles
Doctor of Philosophy (PhD), Washington State University
01/2013
Handle:
https://hdl.handle.net/2376/118009
Abstract
Packaging of eukaryotic DNA into chromatin presents a significant accessibility barrier to proteins that carry out vital DNA-templated processes. Irrespective of this structurally repressive environment, DNA repair must take place to preserve genomic integrity. Proteins overcome this barrier with the assistance of various mechanisms that are capable of inducing changes in chromatin structure, globally and locally. Understanding how these changes in chromatin structure influence DNA accessibility to DNA repair proteins will help identity the parameters and mechanisms that are necessary for efficient repair. Base excision repair (BER) is a major repair pathway primarily responsible for recognition and removal of chemically modified bases. To test BER activity at the primary level of chromatin hierarchy, we have developed an in vitro system where DNA lesions have been rotationally and translationally positioned along the DNA of nucleosome core particles (NCPs). We have tested the ability of purified UDG/APE1 and Pol β to remove their respective lesions on these different nucleosomal substrates. Our results suggest that efficiency of BER in NCPs is greatly dependent not only on the rotational and translational position of the lesion, but also on the structural and/or mechanistic requirements for catalysis, which are enzyme-specific. However, in general, DNA lesions outwardly oriented and farther away from the dyad are most efficiently repaired by both UDG/APE1 and Pol β. These observations hold in the presence of the specific DNA damage-induced histone modifications H3K14Ac and H3K56Ac. This suggests that acetylation at these sites alone does not significantly change the structure and/or unwrapping dynamics of NCPs to override the constraints imposed by the rotational and translational position of the DNA lesions. However, H3K14Ac and H3K56Ac, paradoxically, decrease the gap-filling activity of Pol β near the dyad and have no effect on the removal of uracil. Taken together, our results suggest that the accessibility barrier imposed by the histones on BER enzymes at specific sites may be enzyme-specific and possibly driven by local structural features. Because of this imbalance in repair efficiency, factors that protect BER intermediates or induce changes in NCPs may be necessary for efficient completion of BER in chromatin.
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Details
- Title
- Regulation of Base Excision Repair in Translationally and Rotationally Positioned DNA Lesions in Nucleosome Core Particles
- Creators
- Yesenia Rodriguez
- Contributors
- Michael J Smerdon (Advisor)Margaret E Black (Committee Member)John J Wyrick (Committee Member)Gregory MK Poon (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Pharmaceutical Sciences, Department of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 198
- Identifiers
- 99900581738701842
- Language
- English
- Resource Type
- Dissertation