Examining Kinetic and Thermodynamic DNA Destabilization Caused by the cis-syn Thymine Dimer Lesion Using Small Molecule Probes
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2011-02-16
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Abstract
DNA alternates through continuous cycles of mutation and repair. When DNA is exposed to UV radiation, adjacent thymines within a strand covalently link together, forming a cyclobutane ring through saturation of the 5, 6 double bonds. This lesion is known as a 6,4 cis-syn thymine dimer. Thymine dimers significantly perturb the structure of DNA by kinking the backbone by as much as 30 degrees. These helix-distorting lesions are bulky and can affect the binding of polymerases and sequence-specific proteins.
Repair of DNA lesions can proceed through two main pathways, base excision repair (BER), which repairs small lesions on DNA, and nucleotide excision repair (NER), which repairs large sequences of mutations. The BER pathway begins with an enzyme extruding the damaged base from the double-stranded DNA and then removing the mutated base by breaking the glycosidic bond to the sugar-phosphate backbone. After the excision of the base, the DNA can be reformed through repair synthesis of DNA and DNA ligation.
The process of how a mutation is repaired is understood; however, the process of how the enzyme finds the mutated base within the genome is still unclear. I hypothesized that damaged bases are kinetically and thermodynamically unstable, making them easier to find. I approached this problem using chemical probes to examine the mutation site and the bases around the mutation. Information about the chemical accessibility of the adjacent bases could lead to a clearer picture of how an enzyme finds a mutation. Thus, base-specific reactions were used as chemical probes to examine bases around a thymine dimer mutation. Chemical probes include piperidine formidate, hydrazine, dimethyl sulfate, and potassium permanganate. Using chemical probes to examine the relative base reactivity of unmodified DNA and mutated thymine dimer DNA, quantitative results of DNA destabilization by a thymine dimer have been determined. In particular, the KMnO4 probe, which reacts at only thymine bases, has produced significantly greater overall thymine reactivity on the thymine dimer DNA (AMM1 TT) in comparison to the unmodified DNA (AMM1). Using KMnO4 to probe the complementary strand duplexed with AMM1 and AMM1 TT, the complementary strand duplexed with AMM1 TT was significantly more reactive towards the chemical probe than the AMM1 duplex. This result indicates that a thymine dimer-containing DNA strand makes a double helix more structurally destabilized. Through quantitative base destabilization research, more information can be obtained on the mutagenic characteristics, structural instability, removal and repair of DNA mutations.
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DNA