Date of Award

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Molecular Genetics

First Advisor

Pederson, David

Abstract

ABSTRACT DNA in the eukaryotic nucleus is complexed with histone and non-histone proteins into chromatin. Nucleosomes, the basic repeating unit of chromatin, not only package DNA but are also intimately involved the regulation of gene expression. All DNA transactions including replication, transcription, recombination and repair take place in such a chromatin environment. Access to packaged nucleosomal DNA in vivo is mediated at least in part by protein complexes that modify or remodel chromatin. Buried sequences in nucleosomes can also transiently become accessible to DNA binding proteins during cycles of partial, reversible unwrapping of nucleosomal DNA from the histone octamer. We have investigated the ability of the human, bifunctional DNA glycosylase, endonuclease III (hNTH1), to initiate base excision repair (BER) of discretely positioned oxidative lesions in model nucleosomes. hNTH1 was able to process a thymine glycol (Tg) lesion almost as efficiently as naked DNA, when the minor groove of the lesion faced away from the histone octamer. Lesion processing did not require or result in detectable nucleosome disruption, as assayed in gel mobility-shift experiments. Instead, hNTH1 formed a slower migrating enzyme-nucleosome ternary complex that was found to contain processed DNA. Processing of an inward-facing Tg residue located just 5 bp away from the outward-facing lesion was much reduced and processing of a sterically occluded Tg residue positioned closer to the dyad center of the nucleosome was even more reduced. Notably, processing of both inward-facing lesions was found to increase as a function of enzyme concentration. Restriction enzyme protection studies indicated that access to these inward-facing lesions did not entail nucleosomal translocation or sliding. Collectively, these observations are consistent with a model in which hNTH1 binds to lesions during cycles of reversible, partial unwrapping of nucleosomal DNA from the histone octamer core. To further investigate this partial unwrapping hypothesis, we studied the kinetics of hNTH1 processing of sterically occluded lesions in greater detail. Our results suggest that efficiency of processing of inward-facing lesions is a function of both DNA unwrapping and rewrapping rates, and enzyme affinity for the lesion. In addition, we determined that APE1 which catalyzes the second step in BER, exhibited an increasing capacity to process inward-facing furan residues as its concentration was increased. Thus as with hNTH1, we hypothesize that APE1 can capture occluded furan residues during cycles of partial DNA unwrapping. We propose that cellular regulatory factors benefit from this intrinsic, periodic exposure of nucleosomal DNA exposure in vivo, which may be amplified by the downstream recruitment of remodeling and / or modifying proteins to facilitate DNA transactions in the cell.

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