Impact of sugar pucker on base pair and mispair stability.

The selection of nucleoside triphosphates by a polymerase is controlled by several energetic and structural features, including base pairing geometry as well as sugar structure and conformation. Whereas base pairing has been considered exhaustively, substantially less is known about the role of sugar modifications for both nucleotide incorporation and primer extension. In this study, we synthesized oligonucleotides containing 2'-fluoro-modified nucleosides with constrained sugar pucker in an internucleotide position and, for the first time, at a primer 3'-end.

Chemical decomposition of 5-aza-2'-deoxycytidine (Decitabine): kinetic analyses and identification of products by NMR, HPLC, and mass spectrometry.

The nucleoside analogue 5-aza-2'-deoxycytidine (Decitabine, DAC) is one of several drugs in clinical use that inhibit DNA methyltransferases, leading to a decrease of 5-methylcytosine in newly replicated DNA and subsequent transcriptional activation of genes silenced by cytosine methylation. In addition to methyltransferase inhibition, DAC has demonstrated toxicity and potential mutagenicity, and can induce a DNA-repair response. The mechanisms accounting for these events are not well understood.

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation.

Measurement of the incorporation and repair of exogenous 5-hydroxymethyl-2'-deoxyuridine in human cells in culture using gas chromatography-negative chemical ionization-mass spectrometry.

The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage.

5-halogenated pyrimidine lesions within a CpG sequence context mimic 5-methylcytosine by enhancing the binding of the methyl-CpG-binding domain of methyl-CpG-binding protein 2 (MeCP2).

Perturbations in cytosine methylation signals are observed in the majority of human tumors; however, it is as yet unknown how methylation patterns become altered. Epigenetic changes can result in the activation of transforming genes as well as in the silencing of tumor suppressor genes. We report that methyl-CpG-binding proteins (MBPs), specific for methyl-CpG dinucleotides, bind with high affinity to halogenated pyrimidine lesions, previously shown to result from peroxidase-mediated inflammatory processes.

Synthesis and characterization of oligonucleotides containing 5-chlorocytosine.

Recent studies have shown that reactive chlorine species, derived from myeloperoxidase-mediated inflammation responses, can modify DNA bases, generating 5-chloropyrimidines. The chlorinated adducts could be mutagenic or perturb DNA-protein interactions; however, the biological significance of these adducts is as yet unknown. We report here a method for the synthesis of 5-chlorocytosine- (ClC-) containing oligonucleotides that will be used in subsequent biochemical and biophysical studies to determine the consequences of pyrimidine chlorination.

DNA ligases ensure fidelity by interrogating minor groove contacts.

DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3'-hydroxyl and 5'-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3' side of the ligase junction, but tolerant of mispairs on the 5' side.

5-Formyluracil-induced perturbations of DNA function.

Oxidation of the thymine methyl group can generate 5-formyluracil (FoU), which is known to be both mutagenic and chemically unstable in DNA. Synthetic oligonucleotides containing FoU at defined sites have been prepared to investigate potential mechanisms by which FoU might perturb DNA function. The half-life of the glycosidic bond of an FoU residue in single-stranded DNA under physiological conditions of temperature and pH is estimated to be approximately 148 days, orders of magnitude shorter than the parent pyrimidine, thymine.

Repair of the mutagenic DNA oxidation product, 5-formyluracil.

The oxidation of the thymine methyl group can generate 5-formyluracil (FoU). Template FoU residues are known to miscode, generating base substitution mutations. The repair of the FoU lesion is therefore important in minimizing mutations induced by DNA oxidation.

Influence of local duplex stability and N6-methyladenine on uracil recognition by mismatch-specific uracil-DNA glycosylase (Mug).

To maintain genomic integrity, DNA repair enzymes continually remove damaged bases and lesions resulting from endogenous and exogenous processes. These repair enzymes must distinguish damaged bases from normal bases to prevent the inadvertent removal of normal bases, which would promote genomic instability. The mechanisms by which this high level of specificity is accomplished are as yet unresolved.

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

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.

Substrate recognition by a family of uracil-DNA glycosylases: UNG, MUG, and TDG.

In response to continuous hydrolytic and oxidative DNA damage, cells of all organisms have a complex network of repair systems that recognize, remove, and rebuild the injured sites. Damaged pyrimidines are generally removed by glycosylases that must scan the entire genome to locate lesions with sufficient fidelity to selectively remove the damage without inadvertent removal of normal bases. We report here studies conducted with a series of base analogues designed to test mechanisms of base recognition suggested by structural studies of glycosylase complexes.

Endogenous DNA lesions can inhibit the binding of the AP-1 (c-Jun) transcription factor.

The repair of DNA damage, caused by both endogenous and exogenous sources, is necessary to remove lesions that either miscode or block DNA or RNA polymerases. We propose that damage also must be repaired to maintain sequence-specific DNA-protein interactions. In this paper, we have systematically studied two lesions that interfere with one important DNA landmark, the thymine methyl group.

Characterization of the substrate specificity of a human 5-hydroxymethyluracil glycosylase activity.

The oxidation of pyrimidine 5-methyl groups, derived from either thymine or 5-methylcytosine, can generate 5-hydroxymethyluracil (HmU) in DNA. An activity from HeLa cells that removes 5-hydroxymethyluracil (HmU) from DNA has been partially purified and characterized using a battery of oligonucleotides containing modified bases. This partially purified activity preferentially removes HmU mispaired with guanine.