Inflammation-mediated reactive molecules can result in an array of oxidized and halogenated DNA-damage products, including 5-chlorocytosine ( ClC). Previous studies have shown that ClC can mimic 5-methylcytosine (mC) and act as a fraudulent epigenetic signal, promoting the methylation of previously unmethylated DNA sequences. Although the 5-halouracils are good substrates for base-excision repair, no repair activity has yet been identified for ClC. Because of the apparent biochemical similarities of mC and ClC, we have investigated the effects of mC and ClC substitution on oligonucleotide structure and dynamics. In this study, we have constructed oligonucleotide duplexes containing C, ClC, and mC within a CpG dinucleotide. The thermal and thermodynamic stability of these duplexes were found to be experimentally indistinguishable. Crystallographic structures of duplex oligonucleotides containing mC or ClC were determined to 1.2 and 1.9 Å resolution, respectively. Both duplexes are B-form and are superimposable on a previously determined structure of a cytosine-containing duplex with a rmsd of approximately 0.25 Å. NMR solution studies indicate that all duplexes containing cytosine or the cytosine analogues are normal B-form and that no structural perturbations are observed surrounding the site of each substitution. The magnitude of the base-stacking-induced upfield shifts for nonexchangeable base proton resonances are similar for each of the duplexes examined, indicating that neither mC nor ClC significantly alter base-stacking interactions. The ClC analogue is paired with G in an apparently normal geometry; however, the G-imino proton of the ClC-G base pair resonates to higher field relative to mC-G or C-G, indicating a weaker imino hydrogen bond. Using selective 15N-enrichment and isotope-edited NMR, we observe that the amino group of ClC rotates at roughly half of the rate of the corresponding amino groups of the C-G and mC-G base pairs. The altered chemical shifts of hydrogen-bonding proton resonances for the ClC-G base pair as well as the slower rotation of the ClC amino group can be attributed to the electron-withdrawing inductive property of the 5-chloro substituent. The apparent similarity of duplexes containing mC and ClC demonstrated here is in accord with results of previous biochemical studies and further suggests that ClC is likely to be an unusually persistent form of DNA damage.
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