Artur Burdzy

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. Emerging data suggest that the initial binding of MBPs to methyl-CpG sequences may be a seeding event that recruits chromatin-modifying enzymes and DNA methyltransferase, initiating a cascade of events that result in gene silencing. MBD4, a protein with both methyl-binding and glycosylase activity demonstrated repair activity against a series of 5-substituted pyrimidines, with the greatest efficiency against 5-chlorouracil, but undetectable activity against 5-chlorocytosine. The data presented here suggest that halogenated pyrimidine damage products can potentially accumulate and mimic endogenous methylation signals.

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. We have studied the repair of FoU in synthetic oligonucleotides when paired with A and G. In E. coli cell extract, the repair of FoU is four orders of magnitude lower than the repair of U and is similar for both FoU:A and FoU:G base pairs. In HeLa nuclear extract, the repair of FoU:A is similarly four orders of magnitude lower than the repair of uracil, although the FoU:G lesion is repaired 10 times more efficiently than FoU:A. The FoU:G lesion is shown to be repaired by E. coli mismatch uracil DNA glycosylase (Mug), thermophile mismatch thymine DNA glycosylase (Tdg), mouse mismatch thymine DNA glycosylase (mTDG) and human methyl-CpG-binding thymine DNA glycosylase (MBD4), whereas the FoU:A lesion is repaired only by Mug and mTDG. The repair of FoU relative to the other pyrimidines examined here in human cell extract differs from the substrate preferences of the known glycosylases, suggesting that additional, and as yet unidentified glycosylases exist in human cells to repair the FoU lesion. Indeed, as observed in HeLa nuclear extract, the repair of mispaired FoU derived from misincorporation of dGMP across from template FoU could promote rather than minimize mutagenesis. The pathways by which this important lesion is repaired in human cells are as yet unexplained, and are likely to be complex.

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. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (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 nonstandard 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 use.

105 MEASURING TOXICITY, INCORPORATION, AND REPAIR OF 5-HYDROXYMETHYL-2′-DEOXYURIDINE IN CELLS.

5-Hydroxymethyl-2′-deoxyuridine (HmdU) is among the most common endogenous oxidative DNA lesions and is repaired by more than one DNA glycosylase in human cells. This DNA-repair pathway may be exploited for use in cancer chemotherapy. Exogenous delivery of HmdU to cells in culture leads to its incorporation into the DNA and subsequent repair, resulting in death by apoptosis if the HmdU load is too heavy for the cell. Both incorporation into the DNA and repair of HmdU out of the DNA are necessary for cell death to occur. To measure the incorporation into and subsequent repair of HmdU out of the DNA of cells in culture, we have developed a highly sensitive and specific method using stable isotope dilution and gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) for measuring HmdU levels found in DNA. Using the osteosarcoma cell line U2OS as a model system, we treat cells with HmdU, harvest cells, and isolate DNA. Hydrolysis of the DNA with formic acid releases the free bases, and we convert the base 5-hydroxymethyluracil (HmU) into its methyl ester, 5-methoxymethyluracil, subsequently derivatizing with 3,5-bis(trifluoromethyl)benzyl bromide to yield a stable compound. When analyzing trace levels of HmdU deoxynucleoside standard, this derivatization process yields a detection limit of approximately 500 attomoles (5 × 10−16 mol) on column. We demonstrate in U2OS cells that toxicity of exogenously delivered HmdU is proportional to its incorporation into the DNA, and we show that repair of HmdU out of the DNA is occurring. Application of our method in cell lines with varying sensitivities to HmdU treatment will allow us to gain further insight into the mechanism of and resistance to this potential chemotherapeutic agent.