Date of Award

5-26-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

G-quadruplexes are four-stranded structures of DNA composed of G-quartets that have been proposed to play an extremely important role in replication, transcription, and translation. It has, however, been extremely difficult to unequivocally demonstrate that G-quadruplexes form in living cells due to the lack of probes that would allow for their unambiguous detection and location without disrupting the DNA or the cellular environment. To circumvent these problems, it was proposed that DNA itself could be used as an intrinsic photoprobe for certain classes of G-quadruplex structures and other non-B DNA conformations in vivo. Recently, we discovered that UVB irradiation of human telomeric DNA and various other G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, results in a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). In this thesis, we explore the proposal that anti CPD formation could be used as an intrinsic photoprobe for certain classes of G-quadruplex structures and other non-B DNA conformations in vivo. The idea would be to use a pulse of UVB light to irreversibly trap the non-B DNA structures via anti CPD formation without perturbing the dynamics of the DNA or the cell in the process, after which the anti CPDs could be mapped to provide unambiguous evidence for the formation and location of these structures in vivo As a first step towards this goal, we report the development of radioactive post- and pre-labeling assays for the detection of nonadjacent photoproducts and show its ability to detect the trans,anti T=T CPD that forms in human telomeric DNA. We do so by making use of the enzyme snake venom phosphodiesterase (SVP) to degrade the trans,anti-CPD to a tetramer (pT)pT=(pT)pT, that after dephosphorylation with calf intestinal phosphodiesterase and rephosphorylation with kinase and [32P]-ATP produces a fragment of DNA that can be analyzed by high resolution gel electrophoresis with a fmol lower limit of detection. In the pre-labeling assay, radiolabeled phosphates are introduced into anti T=T CPD forming sites by ligation of 5’-end-labeled oligodeoxynucleotides and only require treatment with SVP. We also demonstrate that the assays can detect the stereoisomeric cis,anti T=T CPD. To determine the scope and limitations of the post-labeling assay method to detect non-adjacent CPDs, we use it to investigate the photochemistry of a number of biologically relevant non-B secondary structures. We report the detection of non-adjacent CPDs under conditions that were previously determined by the NP1-MS assay to produce these photoproducts in insignificant amounts, as well as in structures not expected to form anti CPDs. We show that photoreversal of the CPDs with 254 nm light can be used distinguish DNA photocrosslinked by non-adjacent CPDs from non-photoreversible DNA photoproducts and from partially degraded DNA. We show that the post-labeling assay can be used to corroborate and complement enzyme-coupled mass spectrometry assays resulting in a more complete understanding of the photoreactivity of non-B secondary structures.

Language

English (en)

Chair and Committee

John-Stephen Taylor

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