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 ((Cl)C). Previous studies have shown that (Cl)C can mimic 5-methylcytosine ((m)C) 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 (Cl)C. Because of the apparent biochemical similarities of (m)C and (Cl)C, we have investigated the effects of (m)C and (Cl)C substitution on oligonucleotide structure and dynamics. In this study, we have constructed oligonucleotide duplexes containing C, (Cl)C, and (m)C within a CpG dinucleotide. The thermal and thermodynamic stability of these duplexes were found to be experimentally indistinguishable. Crystallographic structures of duplex oligonucleotides containing (m)C or (Cl)C 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 (m)C nor (Cl)C significantly alter base-stacking interactions. The (Cl)C analogue is paired with G in an apparently normal geometry; however, the G-imino proton of the (Cl)C-G base pair resonates to higher field relative to (m)C-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 (Cl)C rotates at roughly half of the rate of the corresponding amino groups of the C-G and (m)C-G base pairs. The altered chemical shifts of hydrogen-bonding proton resonances for the (Cl)C-G base pair as well as the slower rotation of the (Cl)C amino group can be attributed to the electron-withdrawing inductive property of the 5-chloro substituent. The apparent similarity of duplexes containing (m)C and (Cl)C demonstrated here is in accord with results of previous biochemical studies and further suggests that (Cl)C is likely to be an unusually persistent form of DNA damage.

Figure 1

(a) Reaction scheme for the synthesis of ¹⁵N labeled oligonucleotide. (b) The 12mer sequence and the oligonucleotides synthesized in this study. (c) The chemical structure of the G-C base pair. H_a is the Watson-Crick hydrogen and H_b is non-Watson-Crick hydrogen.

Figure 2

Normalized melting profiles at 260 nm of 28 μM oligonucleotides in presence of 10 mM sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA at pH 7.0.

Figure 3

Structure of the Dickerson-Drew dodecamer with a modified base at position 3. A) Superposition of ^mC (green) and ^ClC (blue) containing duplex with native C dodecamer (gray). B) Difference map between the ^ClC and C dodecamers (Fo^ClC-Fc^C, Φ^ClC ) revealing electron density around Cl on ^ClC at position 3. C) Electron density (2Fo-Fc) for the ^ClC-containing dodecamer. Fo is the observed electron density map and Fc is the calculated electron density map. The structure factors and coordinates of the ^ClC and ^mC-containing dodecamers are deposited to Protein Data Bank with accession number 4MGW and 4MKW, respectively.

Figure 4

¹H NMR spectra in the imino region of the [4-¹⁵N] labeled (X3 position, Figure 1) oligonucleotides (a) ^mC3/C9, (b) C3/C9 and (c) ^ClC3/C9 in the presence of 10% D₂O, 100 mM NaCl, 10 mM sodium phosphate and 0.2 mM EDTA at pH 7 and 300K.

Figure 5

(a) ¹H NMR spectrum (unedited) of the [4-¹⁵N] ^ClC3/C9 oligo. (b-d) ¹⁵N edited proton spectrum of ¹⁵N labelled oligonucleotides in the presence of 10% D₂O, 100 mM NaCl, 10 mM sodium phosphate and 0.2 mM EDTA at pH 7 and 300K. ¹⁵N label is indicated in the structure by the asterisk.

Figure 6

Temperature dependent line width studies of G10 imino protons of C3/C9, ^mC3/C9 and ^ClC3/C9 oligonucleotide in the presence of 10% D₂O, 100 mM NaCl, 10 mM sodium phosphate and 0.2 mM EDTA at pH 7 and 300K.

Figure 7

(a) Proton chemical shifts of the G imino protons of the X3-G10 base pair of the oligonucleotide versus the pK_a of the corresponding nucleoside at pH 7, where X3 is ^mC, C or ^ClC. (b) Amino group rotation rate as a function of the Hammett σ_m parameter of the 5-substituent. Closed circles are for the X3/C9 oligo whereas open circles are for the X3/^mC9 oligo.