Skin cancer remains the most prevalent form of cancer in the United States, with about 1 in 5 Americans contracting basal and squamous skin cancers in their lifetime. Mutation hotspots in tumor suppressor genes in these cancers are C to T and CC to TT mutations that occur mainly at methylated CpG sites that are also hotspots for cyclobutane pyrimidine dimer (CPD) formation. These mutations appear to arise from the deamination of the C's and 5-methylC's (mC) in the CPDs followed by error-free bypass by the DNA damage bypass polymerase eta. What is not well understood, or appreciated, however, is how chromatin structure modulates the rate of deamination and hence the mutagenic potential of mC-containing CPDs, or the epigenetic consequences of CPD formation at methylated CpG sites. Furthermore, the photochemistry of telomeres is largely unknown, in spite of their importance in maintaining genome stability. The major focus of this grant will therefore be to elucidate the structure-activity relationships of photoproduct formation and deamination in nucleosomes and telomeres with regard to their mutagenic and epigenetic consequences. Specifically we propose: (1) To test the hypothesis that chromatin structure can greatly affect the deamination rate and hence the mutagenicity of C/mC containing CPDs, by determining their rate of formation, deamination and repair as a function of rotational and translational position in nucleosome core particles, nucleosomal arrays, and in human cells. We will also determine the physical basis for the modulation of CPD deamination rate by nucleosomes, and the stability and dynamics of nucleosomes containing singly and doubly deaminated CPDs as a function of rotational and translational position in relation to DNA excision repair. (2) To test the hypothesis that error-free bypass of undeaminated mC-containing CPDs contributes to the progression to skin cancer through demethylation of CpG sites, by determining the ability of CPDs to inhibit DNMT1 catalyzed methylation of a complementary strand in vitro, in cell-free extracts, and in human cells. We will also investigate the ability of methyC-binding proteins to enhance the formation of methylC-containing CPDs and prevent their demethylation, thereby promoting the demethylation pathway in preference to the deamination- bypass mutational pathway. (3) To test the hypothesis that photoproduct formation in telomeres may be unusual and could contribute to skin cancer induction, by determining the structure of the photoproducts that are induced by UVB light in telomeric DNA in human cells and compare with those formed in the presence of telomere DNA binding proteins. We will also determine the rates of deamination of C-containing CPDs that would be predicted to lead to telomere mutagenesis and destabilization.
Knowledge gained through this research will provide a better understanding of the mechanisms by which skin cancer arises, and may led to better risk assessment and preventative strategies, as well as diagnostic methods, prognosis, and therapeutic strategies.
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