Emerging data suggests that the development of cancer in man is a multi-step process driven by not only genetic damage but also epigenetic changes. Perturbations in epigenetic signals can result in the loss of tumor suppressor expression or inappropriate oncogene expression. The molecular basis of epigenetic control involves the enzymatic methylation of cytosine residues in CpG dinucleotides and proteins that selectively bind to methylated DNA sequences, initiating a cascade of interacting proteins that alter local chromatin structure and transcriptional activity. While the list of epigenetic perturbations in human tumors grows daily, the mechanisms by which epigenetic signals become scrambled in tumor development are largely unexplored. In the initial funding period, we discovered that oxidative damage and halogenation arising from inflammatory damage could result in loss of DNA methylation. This competing renewal application focuses on exploration of the mechanisms whereby halopyrimidines could alter epigenetic signals. In the five aims of the current application, we propose to 1) further map critical contact points between methyl-binding proteins and their DNA-binding sites with nucleoside analogs and purified methyl-binding proteins in gel electrophoretic mobility shift assays, 2) examine the structure, dynamics and coding potential of 5-chlorocytosine (5CIC) and 5-chlorouracil (5CIU)-containing oligonucleotides using high field isotope edited NMR and polymerase extension assays, 3) examine the reactivity of cytosine residues in a CpG dinucleotide toward chlorination reactions using a recently developed mass-tagging assay, 4) examine the capacity of 5-chlorpyrimidines to direct methylase-mediated methylation or to inhibit methylation, and 5) to examine the capacity of 5CIC residues to inhibit transcription and induce heritable epigenetic changes in mammalian cells. This proposal's results will likely shed important new light on mechanisms by which DNA damage could result in heritable epigenetic alterations critical in the development of many human tumors.
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