Barlev, Adam - DNA Repair by DNA with Visible Light: Investigations and Implications...

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Term: 
Fall 2017
Degree: 
Ph.D.
Degree type: 
Thesis
Department: 
Department of Chemistry
Faculty: 
Science
Senior supervisor: 
Dipankar Sen
Thesis title: 
DNA Repair by DNA with Visible Light: Investigations and Implications
Given Names: 
Adam
Surname: 
Barlev
Abstract: 
The DNAzyme UV1C was selected previously on the basis of its ability to utilize UV-B light to catalyze the repair of a cis-syn cyclobutane thymine dimer in which no phosphodiester linkage exists between the dimerized thymines. Systematic replacement of each of nine guanines in and around the active site by the guanine analog 6-MI allowed the expansion of the photocatalytic cross section throughout the UV-A and to the edge of the visible. The behaviour of these mutants fell into 3 classes. In one class, replacement of guanines in the quadruplex did not disrupt the wild-type activity. In another class, quadruplex positions, when replaced with 6-MI, led to a decrease in activity in the UV-B but new activity in the UV-A, providing strong evidence for exactly which guanine residues are catalytic in the DNAzyme. Most surprisingly, the G-23 position, thought to be near the active site but not catalytic in UV1C, when replaced with 6-MI, leads to a full retention of activity in the UV-B with the strongest gain of activity in the UV-A. Further modifications to the G-23 position pushed its activity to maximize in the visible, but also ultimately disrupted the quadruplex-dependent activity in the UV-B. While selected against a model thymine dimer substrate, the DNAzyme is also shown to have photocatalytic activity on a bona fide DNA substrate. The continuity of the natural DNA substrate allows us to measure for the first time the effect of the UV1C DNAzyme on the rate of both thymine dimer formation as well as the rate of repair. When compared to double-stranded and single-stranded controls, at its photostationary state, UV1C leads to an overall reduction in fraction of dimerized thymines. Surprisingly, UV1C catalyzes both the repair and formation of thymine dimers in natural DNA, but more slowly than the model substrate that it was selected against. Together, these results shed further light on the emerging field of protein-independent thymine dimer repair. Arguments connecting the self-repair properties of DNA to the RNA world and prebiotic chemistry are offered.
Keywords: 
DNA repair; Photocatalysis; DNAzyme; Thymine Dimer; G-quadruplex; RNA world
Total pages: 
128