Date of Award

Summer 8-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites, and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggest that DNA bending causes the modulation of photoproduct formation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we had developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We had used this system to show that the nucleosome dramatically modulates CPD formation in a T11-tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5’-side of that found in mixed-sequence nucleosomes. We have also shown that an 80-mer minicircle DNA using the same TG motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation. Furthermore, we have used the mono-nucleosome system to show that nucleosome moderately modulates TC CPD deamination in poly (TC)n (n = 6 and 35) tracts that covers almost all seven SHLs in one full nucleosome helix. Unlike a previous study of deamination at TCG sites, the position of maximum CPD deamination rate is shifted 3 nucleotides to the 5’-side of the base pairs whose major groove face the nucleosome.

Language

English (en)

Chair and Committee

John-Stephen Taylor

Committee Members

Michael L. Gross, Jay Ponder, Douglas Chalker, Timothy Wencewicz,

Comments

Permanent URL: https://doi.org/10.7936/K7DR2TWW

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