Differential Stabilities and Sequence-Dependent Base Pair Opening Dynamics of Watson–Crick Base Pairs with 5-Hydroxymethylcytosine, 5-Formylcytosine, or 5-Carboxylcytosine

Abstract

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson–Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5′-CG-3′ sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5′-T8X9G10-3′ sequence of the DDD, were compared. The presence of 5caC at the X9 base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A5:T8, whereas 5caC did not. At the oxidized base pair G4:X9, 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C3:G10. No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G4:X9; each favored Watson–Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N4 exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.

5-Formylcytosine does not change the global structure of DNA

“…The overall reaction involves the enzyme binding to DNA, followed by bending and inserting an amino acid residue via the minor groove to help flip out the desired base, exposing the glycosyl bond for cleavage within the TDG active site. Such a structure in the absence of enzyme would be at a much higher energy than the relaxed B-DNA, and our data and those of Szulik et al 19 indicate that the population of flipped out bases is very low in free f C-DNA. To achieve a productive complex, the flipped-out base must make a large number of interactions with the enzyme to compensate for the local energy of distortion of the duplex.…”

“…This similarity is also the case for f C in RNA which is also A-form, 37 and in a modified Dickerson-Drew dodecamer containing f C which is B-form in the crystal state. 19,21 Furthermore, our NMR data show that in solution these duplexes all adopt the B conformation, and that the differences induced by the f C modification are rather small and local, and unlikely to act as a gross structural recognition feature for TET, 38 TDG 39 or other enzymes. We conclude that there is no compelling evidence for a unique f C-dependent F-DNA structure, nor are the specific hydration patterns in the X-ray structure likely to be relevant for B-form duplexes.…”

“…Thus the incorporation of f C influences the local nucleotide conformation, characterized by a somewhat larger glycosyl torsion angle, and a less pure C2′-endo like sugar pucker, but modest nucleotide-level conformational perturbation is not propagated throughout the helix, and thus does not have a global influence on the structure, in agreement with Szulik et al . 19 The 5-formyl group has a large electronic effect on the nucleobase, as seen in the chemical shift of the f CH6 (ca. 0.9 ppm downfield of C), and in the nearest neighbour nucleotides.…”

A DNA structural alphabet provides new insight into DNA flexibility

“…On average, the confal score improved by 7 after application of the PDB_REDO protocol, but the fraction of reassigned NtC classes varied widely between 0 and 77% in individual structures. The confal values showed significant improvement, for example, in the structure with PDB entry 1d29 (Larsen et al, 1991), while no changes were observed in PDB entry 4i9v (Szulik et al, 2015) and a mixture of increased and lowered confal values were found in PDB entry 5bna (Wing et al, 1984) (Supplementary Table S3). Overall, the PDB_REDO protocol improves the deposited coordinates somewhat, but better molecular templates provided by the NtC classes can constrain the re-refinement more effectively.…”

The Mechanisms of Generation, Recognition, and Erasure of DNA 5-Methylcytosine and Thymine Oxidations

“…Furthermore, the structurally related E. coli mismatch uracil glycosylase (eMUG) can excise 5caC and 5fC as well (74,82). Both 5fC and 5caC exhibit an intrabase hydrogen bond between their formyl or carboxyl oxygen atoms, respectively, and the adjacent cytosine N 4 exocyclic amine nitrogen atom, both in the free form (83) and in the protein-bound form (58) (Fig. 3, h and i, top panels).…”