Comparison of the Structural and Dynamic Effects of 5-Methylcytosine and 5-Chlorocytosine in a CpG Dinucleotide Sequence

Abstract

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. Due to 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 are found to be experimentally indistinguishable. Crystallographic structures of duplex oligonucleotides containing mC and ClC were determined to 1.2 and 1.9 Å, respectively. Both duplexes are B-form and are superimposable on a previously determined structure of a cytosine-containing duplex with RMSD of approximately 0.25 Å. NMR solution studies indicate that all duplexes containing cytosine or the cytosine analogs are normal B-form, and no structural perturbations are observed surrounding the site of each substitution. The magnitude of the base-stacking induced upfield shifts for non-exchangeable base proton resonances are similar for each of the duplexes examined, indicating that neither mC nor ClC significantly alter base stacking interactions. The ClC analog 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 the rate of the corresponding amino groups of the C-G or 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.

Intrinsic mutagenic properties of 5-chlorocytosine: A mechanistic connection between chronic inflammation and cancer

“…In comparison, m5C has a pK a similar to that of cytosine (4.35) (23). The electronic effect of chlorine was shown to be strong enough to influence the energy of the hydrogen bonds involved in basepairing; thus, the estimated base-pairing energy of 5ClC:G is slightly less than the energy of the C:G pair (43). Future studies probing the electronic configuration of 5ClC will be of interest to provide a more comprehensive understanding of the way that the chlorine substituent alters the base-pairing potential of 5ClC.…”

Occurrence, Biological Consequences, and Human Health Relevance of Oxidative Stress-Induced DNA Damage

“…Base-pairing energy of 5-Cl-C:G is only slightly lower than that of the C:G pair, 266 suggesting that 5-Cl-C is likely a persistent DNA lesion. It was recently reported that all families of DNA polymerases predominantly decode 5-Cl-C as C in vitro ; meanwhile, 5-Cl-C, when placed on single-stranded M13 plasmid and replicated in E. coli cells, induced 3–9% C → T transition, which is as mutagenic as 8-oxo-dG in similar assays.…”

Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes

“…In the presence of PEG200, the plot showed a linear relationship. From the slope of the linear relationship, it is possible to evaluate the value of Δ n w , which is the number of water molecules taken up upon formation of a structure [ 56 , 57 ]. The values of Δ n w were estimated to be 96, 105, 108, and 106 for Me0, Me3, Me5, and Me8, respectively, corresponding to an uptake of 9.6, 10.5, 10.8, and 10.6 water molecules per base pair, respectively.…”

“…Spectroscopic studies have shown that the overall structure of the DNA duplex is not largely affected by cytosine methylation [ 26 , 56 , 57 , 58 ]. However, it has been reported using single-molecule force spectroscopy that cytosine methylation has a significant effect on the separation of a duplex [ 59 ].…”