Author's Department/Program
Chemistry
Language
English (en)
Date of Award
1-1-2010
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Chair and Committee
John-Stephen Taylor
Abstract
Exposure of cells to ultraviolet light results in the formation of DNA photoproducts, many of which have been linked to skin cancer. The majority of these photoproducts arise from a photoreaction between two pyrimidines. Irradiation of double stranded B form DNA under physiological conditions mainly produces adjacent dimers. Nonadjacent photodimers of both intrastrand and interstrand types are much rarer. Intrastrand-type nonadjacent dimers form when one or more nucleotides between two pyrimidines become extrahelical owing to the formation of single strand DNA or a slipped structure. This allows the two pyrimidines to photodimerize in a colinear arrangement, as if the intervening sequence was not present. Enzyme digestion, chemical hydrolysis, high performance liquid chromatography: HPLC) coupled with mass spectrometry: MS) have been been powerful tools for identification and characterization of DNA photoproducts. Using these tools we discovered an unusual nonadjacent thymine dimer product formed between two T's with 5 nucleotides apart in a single-standed 14-mer ODN sequence. This novel and unexpected photoproduct arises from an interstrand-type reaction due to an unusual higher order folded DNA structure. This finding led us to investigate the photochemistry of biologically relevant G-quadruplexes, which have high biological relevance in telomers. We studied the consequences of UV irradiation of G-quadruplex structures of a 22-mer telomeric sequence by utilizing site-specifically modified DNA, HPLC, CD, and nuclease P1: NP1) coupled with MS. A large number of specific anti thymine dimers were formed between two T's located in different loops of the telomeric G-quadruplexes. If these unique nonadjacent anti thymine dimer photoproducts also form in vivo, they may constitute a previously unrecognized type of DNA photodamage that interferes with telomere replication and present a unique challenge to DNA repair. We also describe the characterization of a new photoproduct mCA*, whose deamination product has the same structure as the well-known TA* photoproduct. The structure proof was done with a combination of NP1 digestion, HPLC correlation, and MSn techniques. A second part of the thesis deals with a DNA-binding protein. Human apurinic/apyrimidinic endonuclease 1: hApe1) contributes to DNA repair by cleaving apurinic/apyrimidinic sites of damaged DNA. Additionally, hApe1 is a bifunctional protein functioning as a redox factor that simulates DNA binding activity of several transcription factors that are involved in cancer promotion and progression including AP-1, P53 and others. The underlying mechanism of its redox activity, however, is poorly understood. One proposed mechanism is the disulfide exchange between hApe1 and the transcription factors. It's also hypothesized that hApe1 undergoes conformational changes that allows it to interact with the transcription factors. A quinone derivative E3330,: 2E)-3-[5-(2,3-dimethoxy-o-methyl-1,4-benzoquinoyl)]-2-nonyl-2-propenoic acid, is a specific redox-activity inhibitor for hApe1. We used MS-based methods to probe the structure, dynamics, and interactions of HApe1 with ligands. We used amide hydrogen/deuterium exchange: HDX) and N-ethylmaleimide: NEM) labeling coupled MS analysis to probe the interaction of hApe1 and E3330. Our study suggests there is a minor amount of a locally unfolded form existing in equilibrium with the major folded form of hAp1. This locally unfolded form interacts with E3330 and is stabilized, allowing formation of disulfide bonds: C65-C93, C65-C99, C93-C99) in the redox active domain. A previous cell-based assay suggests that C65 is essential and thatC93 and C99 are also involved in the redox activity of hApe1. Taken together, we propose that the locally unfolded form is responsible for the redox function of hApe1 via a thiol/disulfide mechanism.
Recommended Citation
Su, Dian, "Discovery and Identification of New DNA Photoproducts and Investigation of the Redox Mechanism of Human Apurinic/Apyrimidinic Endonuclease 1 (hApe1) Protein" (2010). All Theses and Dissertations (ETDs). 337.
https://openscholarship.wustl.edu/etd/337
Comments
Permanent URL: http://dx.doi.org/10.7936/K798852S