Cytosine methylation in CpG dinucleotides is believed to be important in gene regulation, and is generally associated with reduced levels of transcription. Methylation-mediated gene silencing involves a series of DNA-protein and protein-protein interactions that begins with the binding of methyl-CpG binding proteins (MBPs) followed by the recruitment of histone-modifying enzymes that together promote chromatin condensation and inactivation. It is widely known that alterations in methylation patterns, and associated gene activities, are often found in human tumors. However, the mechanisms by which methylation patterns are altered are not currently understood. In this paper, we investigate the impact of oxidative damage to a methyl-CpG site on MBP binding by the selective placement of 8-oxoguanine (8-oxoG) and 5-hydroxymethylcytosine (HmC) in a MBP recognition sequence. Duplexes containing these specific modifications were assayed for binding to the methyl-CpG binding domain (MBD) of one member of the MBP family, methyl-CpG binding protein 2 (MeCP2). Our results reveal that oxidation of either a single guanine to 8-oxoG or of a single 5mC to HmC, significantly inhibits binding of the MBD to the oligonucleotide duplex, reducing the binding affinity by at least an order of magnitude. Oxidative damage to DNA could therefore result in heritable, epigenetic changes in chromatin organization.

The nucleoside analog 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. DAC is chemically unstable in aqueous solutions, but there is little consensus between previous reports as to its half-life and corresponding products of decomposition at physiological temperature and pH, potentially confounding studies on its mechanism of action and long-term use in humans. Here we have employed a battery of analytical methods to estimate kinetic rates and to characterize DAC decomposition products under conditions of physiological temperature and pH. Our results indicate that DAC decomposes into a plethora of products, formed by hydrolytic opening and deformylation of the triazine ring, in addition to anomerization and possibly other changes in the sugar ring structure. We also discuss the advantages and problems associated with each analytical method used. The results reported here will facilitate ongoing studies and clinical trials aimed at understanding the mechanisms of action, toxicity, and possible mutagenicity of DAC and related analogs.

No abstract

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. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3' side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson-Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson-Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson-Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3' side of the ligase junction. The NAD+-dependent ligase, Tth, is more sensitive to the DFT analog on the unligated strand whereas the ATP-dependent T4 ligase is more sensitive to substitutions in the template strand. Electrophoretic gel mobility-shift assays demonstrate that the Tth ligase binds poorly to oligonucleotide substrates containing analogs with altered minor groove contacts.

Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of structural 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. In this paper, we describe the application of MALDI-TOF-MS to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with non-standard synthesis, deprotection or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and utilization.