Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Chemistry

Language

English (en)

Date of Award

Winter 1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

John-Stephen A. Taylor

Abstract

C-to-T mutations are a hallmark of UV light and, in humans, occur preferentially at methylated PymCG sites, which are also sites of preferential cyclobutane pyrimidine dimer: CPD) formation. CPDs containing C or 5-methylcytosine: mC) are not stable and spontaneously deaminate to U or T at pH 7 and 37°C over a period of hours or days. Deamination of Cs or mCs in CPDs is highly mutagenic because polymerase ç will faithfully insert A opposite the resulting Us or Ts, thereby producing the observed C to T and CC to TT mutations: the deamination-bypass mechanism). In this thesis, we prepared a CPD of a TmCG site, a known hotspot for C methylation, CPD formation, and UV light-induced C-to-T mutations found in the p53 gene of basal and squamous cell cancers. We show that both yeast and human pol ç could synthesize past the 3'-mC in T=mC CPD in a >99% error-free manner by non-mutagenic insertion of G opposite the mC in the CPD. We also confirmed the error-free but mutagenic insertion of A opposite the resulting deaminated T.

Nucleosomes are the primary structural unit of chromatin in eukaryotic cells. UV preferentially induces the formation of CPDs in nucleosomes at sites where the phosphodiester backbone is positioned away from the histone surface and DNA bending is toward the major groove. Nucleosomes have also been found reduce the rate of nuclear excision repair to different extents depending on specific tissue and cell type. Although the frequency of CPD formation and repair is modestly modulated by its rotational positioning within a nucleosome, the effect of nucleosome rotational positioning on the rate of deamination of mC in a CPD has not been previously studied. In this thesis, we investigated the deamination of CPDs in a chicken erythrocyte nucleosome core particle reconstituted with synthetic DNA. We found that the deamination of a TmC CPD whose sugar phosphate backbone is positioned against the histone core surface decreases by a factor of 4.7, whereas that of a TmC CPD positioned away from the surface increases by a factor of 8.9 when compared with unbound DNA. Considering that formation of the CPD positioned away from the surface is also enhanced by a factor of two, a TmCG site in this position might be expected to have up to an 84-fold higher probability of resulting in a UV-induced mC to T mutation than one positioned against the surface. We also determined the deamination rate for T=mC CPDs in all ten possible nucleosome rotational positions of a full periodic turn on the nucleosome core particle surface, at the same translational position. Three T=mCG CPDs positioned inside deaminated slower than the unbound DNA, whereas the seven positioned outside deaminated faster than the unbound sequence.

Many of researchers have relied on nucleosome core particles isolated directly from organisms for their in vitro studies. Although the isolation process is straightforward, it is generally time-consuming, and may contain modified histones. Recombinant histones offer the possibility to carefully study the structure activity relationships of nucleosome core particles containing DNA photoproducts, and in this thesis, we report the preparation and assembly of a nucleosome core particle from recombinant histones. A histone octamer was prepared from recombinant Xenopus histones expressed and purified from E. coli, based on minor modifications of previously described protocol. We also reconstituted the recombinant histone octamer with our synthetic 147-mer DNA duplex, and demonstrated that it assembled with a specific rotational and translational position.

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Permanent URL: http://dx.doi.org/10.7936/K7DV1GWD

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