Nei-like 1 (NEIL1) excises 5-carboxylcytosine directly and stimulates TDG-mediated 5-formyl and 5-carboxylcytosine excision.

Thymine DNA glycosylase (TDG) and Nei-like 1 (NEIL1) have both been implicated in the base excision repair step of active DNA demethylation. The robust glycosylase activity of TDG on DNA substrates containing 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC) is universally accepted, but the mode of action of NEIL1 is still debated. Based on genetic experiments, it has been suggested that NEIL1 acts redundantly with TDG and excises 5fC and 5caC directly. However, this result has been disputed, and it was suggested instead that NEIL1 is recruited by the monofunctional TDG for the 2'-deoxyribose excision step. Using purified human NEIL1 and its catalytically impaired P2T and E3Q variants as controls, we detect NEIL1 activity on 5caC, but not a 5fC containing dsDNA substrate. We confirm direct NEIL1 TDG binding and NEIL1 mediated 2'-deoxyribose excision downstream of TDG glycosylase activity. NEIL1 acts not only downstream of TDG, but also enhances TDG activity on 5fC or 5caC containing DNA. NEIL1 mediated enhancement of the TDG glycosylase activity is substrate specific and does not occur for dsDNA with a T/G mismatch.

On the role of steric clashes in methylation control of restriction endonuclease activity.

Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not 'fit' into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 Å(3) for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation.

Structural basis of the methylation specificity of R.DpnI.

R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support 'crosstalk' between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity.