THE ELUCIDATION OF THE STRUCTURE AND KINETICS OF A DECAMER DNA DUPLEX CONTAINING A CIS-SYN THYMINE DIMER
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When DNA is exposed to ultraviolet light, in some cases it becomes photo-damaged and results in a cis-syn pyrimidine dimer, referenced as the thymine dimer. Crystallography and NMR studies on photo-damaged DNA report an induction of an unusual locked conformation between the thymine dinucleotides, duplex strain of ~30° towards the major groove to accommodate the thymine dimer, and a loss of aromaticity. However, the entire DNA duplex remains right handed and maintains stacked bases that remain paired with their complementary nucleotide.1 Despite this structural information, the extent to which the dimer could increase the mobility or dynamics of a DNA duplex remain unresolved. The imino protons of the dimer thymines and the flanking bases exhibit sharp peaks in the 1H NMR spectra. NMR studies support the existence of these phenomena in the presence of the thymine dimer. Moreover, these studies demonstrate the formation of stable hydrogen bonds throughout the entire DNA duplex. These results are used to inform the mechanisms by which DNA performs its functions and maintains its structure. The structural changes that occur in the DNA duplex containing a thymine dimer are to a degree that may prevent repair enzymes from recognizing the damaged conformation. This results in an interference during replication and transcription because of the local and global changes to the decamer dimer. Fortunately, some enzymes have developed mechanisms to both recognize and repair cis-syn pyrimidine dimers. Such enzymes are Escherichia coli (E. coli) photolyases and T4EndoV. Additionally, there are general repair enzymes such as human exconuclease and E.coli uvrABC. The further elucidation of the structural and dynamic properties of the thymine dimer will allow for better understanding of the mechanisms of recognition and repair of damaged DNA by this subset of repair enzymes. Using 1D NMR exchange experiments and 2D NMR structural mapping, it is possible to elucidate the structural and dynamic properties of these DNA duplexes upon dimerization. Using 1D NMR, the solution accessibility to the imino protons within the DNA and the kinetic properties of the imino protons was probbed. With sufficiently high catalyst concentration, exchange can occur for each opening event. Our results demonstrate that in the perturbed DNA duplex, the thymine 5’ to the dimer exchanges at rates comparable to the penultimate cytosines and guanines. This informs the positioning of the imino protons that are being exchanged with catalyst, in addition to the non-exchangeable protons interactions with one another and location throughout the duplex.
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