Comparison of the structural and dynamic effects of 5-methylcytosine and 5-chlorocytosine in a CpG dinucleotide sequence

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.