Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA

Abstract

A specific and efficient method is presented for the conversion of 2'-deoxyuridine to thymidine via formation and reduction of the intermediate 5-hydroxymethyl derivative. The method has been used to generate both thymidine and 5-methyl-2'-deoxycytidine containing the stable isotopes 2H, 13C and 15N. Oligodeoxyribonucleotides have been constructed with these mass-tagged bases to investigate sequence-selectivity in hydroxyl radical reactions of pyrimidine methyl groups monitored by mass spectrometry. Studying the reactivity of 5-methylcytosine (5mC) is difficult as the reaction products can deaminate to the corresponding thymine derivatives, making the origin of the reaction products ambiguous. The method reported here can distinguish products derived from 5mC and thymine as well as investigate differences in reactivity for either base in different sequence contexts. The efficiency of formation of 5-hydroxymethyluracil from thymine is observed to be similar in magnitude in two different sequence contexts and when present in a mispair with guanine. The oxidation of 5mC proceeds slightly more efficiently than that of thymine and generates both 5-hydroxymethylcytosine and 5-formylcytosine but not the deaminated products. Thymine glycol is generated by both thymine and 5mC, although with reduced efficiency for 5mC. The method presented here should be widely applicable, enabling the examination of the reactivity of selected bases in DNA.

The Carboxy-Terminal Domain of ROS1 Is Essential for 5-Methylcytosine DNA Glycosylase Activity

“…The four chemically modified forms of cytosine might not be genetically equivalent in terms of base pairing. A strong intramolecular hydrogen bond has been observed between the exocyclic N 4 amino group (NH 2 ) and the carbonyl oxygen (O=C) at ring carbon-5 position of 5fC, in the free nucleoside form 49,50 , and the carboxyl moiety (COO − ) of 5caC in the protein bound form 51 . It was hypothesized that the existence of such an intra-base hydrogen bond would shift the amino-imino equilibrium 48,52,53 , which would enable 5fC and 5caC to form two, instead of three, hydrogen bonds with an opposite guanine, equivalent to a G:T or G:U ‘wobble’ pair.…”

Quantification of oxidative single-base and intrastrand cross-link lesions in unmethylated and CpG-methylated DNA induced by Fenton-type reagents

“…Similar addition of hydroxyl radical to the C5=C6 double bond of mC can lead to the formation of mC glycol, which can deaminate to give thymine glycol (53,54). Because of the lack of isotope-labeled internal standards of thymidine glycol, the formation of this lesion from the mC-bearing strand was not quantified in the present study.…”

“…Because of the lack of isotope-labeled internal standards of thymidine glycol, the formation of this lesion from the mC-bearing strand was not quantified in the present study. In addition, the hydroxyl radical can abstract a hydrogen atom from the methyl group of mC to give the methyl radical of the pyrimidine base, which can further transform to give 5-HmdC and 5-FmdC under aerobic conditions (54). With the above quantification results, it is reasonable to speculate that the total amount of hydroxyl radical-induced major single-base lesions from mC can be several fold higher than those formed from unmethylated cytosine.…”

Apoptosis and DNA Methylation

“…Its importance in epigenetics is that the hydroxymethyl group is suggested to alter the biological properties of methylated DNA. It is worth noting that the methyl groups of both thymine and 5mC are susceptible to oxidation with 5mC being slightly more reactive resulting in the generation of hmC [11]. hmC also presents a new experimental problem, as conventional techniques (with the exception of a hmC specific antibody), cannot distinguish between 5mC and hmC in DNA [12,13].…”