Skin cancer remains the most prevalent form of cancer in America, affecting one in three Americans during their lifetime. The most prevalent mutations induced by sunlight are C to T and CC to TT mutations, and have been principally correlated with cis-syn cyclobutane pyrimidine dimers (CPDs), whose formation is greatly enhanced at sites of CpG methylation. Most of these C to T mutations appear to arise from a deamination bypass mechanism, in which the C or methylC (mC) in a CPD deaminates to U or T which directs the insertion of A by polymerase eta. There is also evidence that suggests that some of these mutations may also arise from a tautomer bypass mechanism in which the C or methylC tautomerizes to the E-imino isomer that resembles a U or T and likewise directs the insertion of A. The principal goal of this proposal is to better understand the DNA photoproduct and polymerase structure-activity relationships involved in the deamination and tautomerization bypass mechanisms for the formation of C to T mutations.
One specific aim i s to develop efficient and stereocontrolled syntheses of T, C, and mC-containing CPDs to facilitate the preparation of substrates for enzymatic, NMR, and crystallographic studies.
A second aim will be to use 15N-labeled CPDs to study H-bonding, tautomerization, and proton exchange of these photoproducts in DNA duplexes.
A third aim will be to determine the effect of pH, buffer, sequence context, DNA conformation, and protein binding on the deamination rates of C and methylC dimers in DNA by enzyme-coupled mass spectrometric, polymerase bypass, and PCR assays.
A fourth aim will be to study the effect of sequence context on the mutagenicity of DNA synthesis past T and C-containing CPDs by pol eta and accessory proteins through the use of competition assays, pre-steady state kinetics and a new serial analysis of mutation spectra (SAMS) assay that we have developed. We will also make use of nucleotide analogs to probe the polymerase active site, H-bonding, and tautomerization state of photoproducts. A fifth aim will be to carry out crystallographic, solid state NMR, mutagenesis, and mass spectrometric assays of ternary complexes of pol eta with T and mC-containing CPDs to understand the structural and mechanistic basis of nucleotide insertion selectivity and efficiency.
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