DNA is constantly being damaged by both endogenous and exogenous sources, e.g. light, oxygen radicals, chemical contaminants, societal habits. This damage, if not immediately repaired, can result in DNA miscoding and mutation, leading to cancer, teratogenesis, cardiovascular problems, and aging. Understanding the chemistry of DNA lesions and the mechanisms by which they miscode are important in considerations of assessing risks involved. In this proposal, we plan to build on previous research in this laboratory to understand two types of DNA damage. (A) We propose to address the hypothesis that two types of DNA adducts are miscoding, namely N7-alkyl guanine and N3-alkyl adenine adducts, both of which have unstable glycosidic bonds and tend to depurinate. However, the rates of depurination are relatively slow and the potential for miscoding exists. We will produce oligonucleotides with stable analogs by using a 2-fluoro isostere approach, which has some precedence, including our own work. The methylated derivatives will be examined first, followed by several other complex adducts known to arise from environmental chemicals. Miscoding will be testing in vitro using a series of bacterial and human DNA polymerases, with analysis using gel electrophoresis and mass spectrometry. (B) Another Aim will test the hypothesis that certain peptide- chemical-DNA crosslinks are miscoding. One aspect will deal with the tripeptide glutathione (GSH), known to form DNA crosslinks in vivo with 1, 2-dibromoethane and butadiene diepoxide, two chemicals of interest. A hypothesis to be addressed is that initial O6-alkylguanine DNA-alkyltransferase (AGT)-DNA crosslinks formed with these two chemicals are processed by cellular proteases to yield peptides that are small enough to be bypassed and also miscode. We will test aspects of the hypothesis and, if it is true, characterize aspects of the crosslinked peptides and how they fit into DNA polymerases that can include them inside their structures. Collectively the information is about the unstable linkage and the cross-linked DNA adducts. These investigations are designed to provide important new information about how some types of DNA damage occur and, most importantly, what their biological consequences are.
Many chemicals cause cancer by binding to the DNA in the cells of the body. When the genetic material (damaged DNA) is miscopied mistakes may occur (mutations) and lead to cancer. The goals of this project involve understanding how two kinds of damage can cause mutations that lead to diseases.
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