Dissertation
Excision Repair and DNA Damage Tolerance in the Context of Nucleosomes
Doctor of Philosophy (PhD), Washington State University
01/2015
Handle:
https://hdl.handle.net/2376/111053
Abstract
Chromatin is occlusive to DNA damage response factors that process DNA lesions to reduce mutations and forego programmed cell death; paradoxically, chromatin is essential for cell viability because it is required for the compaction of genomic DNA in the nucleus and modulation of metabolic processes. The primary structural component of chromatin is the nucleosome, which consists of ~147 base pairs of DNA wrapped around a histone octamer. To understand how histones contribute to DNA damage response, we analyzed amino-terminal tail (N-tail) deletions of all four canonical histones, both individually and in combination, in the budding yeast Saccharomyces cerevisiae. We found that combinatorial N-tail deletions of histones H2A and H3 (tH2A:tH3) sensitize yeast cells to the DNA alkylating agent methyl methanesulfonate (MMS), and epitasis analyses indicate that multiple DNA damage response pathways are involved in this MMS sensitivity phenotype. Interestingly, the tH2A:tH3 mutant is deficient in base excision repair (BER), a repair pathway with two subpathways that differ in repair patch sizes. We developed an in vitro assay to differentiate between these subpathways and showed that DNA lesions located in the nucleosome core are preferentially repaired by DNA polymerase β that insert short repair patches. Additionally, the tH2A:tH3 mutant was shown to be deficient in nucleotide excision repair (NER), a repair pathway for DNA helix-distorting lesions, such as those induced by UV light. NER also has two subpathways differentiated by the presence or absence of the transcription machinery. We found that transcription-coupled NER requires the removal of the posttranslational ubiquitin moiety from H2B at lysine residue 123 to destabilize chromatin structure and allow for accessibility by repair enzymes. Altogether, our data show that even though chromatin is inhibitory to DNA damage response factors, chromatin provides signals to promote different DNA damage response pathways and subpathways, including those of BER and NER.
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Details
- Title
- Excision Repair and DNA Damage Tolerance in the Context of Nucleosomes
- Creators
- Rithy Meas
- Contributors
- Michael J Smerdon (Advisor)John J Wyrick (Advisor)Michael E Konkel (Committee Member)Lisa M Gloss (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 220
- Identifiers
- 99900581637801842
- Language
- English
- Resource Type
- Dissertation