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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA040463-25
Application #
7646317
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Okano, Paul
Project Start
1985-07-01
Project End
2011-07-31
Budget Start
2009-08-01
Budget End
2010-07-31
Support Year
25
Fiscal Year
2009
Total Cost
$269,587
Indirect Cost
Name
Washington University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Smith-Carpenter, Jillian E; Taylor, John-Stephen (2018) Photocrosslinking of G-Quadruplex-Forming Sequences found in Human Promoters. Photochem Photobiol :
Lu, Chen; Smith-Carpenter, Jillian E; Taylor, John-Stephen A (2018) Evidence for Reverse Hoogsteen Hairpin Intermediates in the Photocrosslinking of Human Telomeric DNA Sequences. Photochem Photobiol 94:685-697
Wang, Kesai; Taylor, John-Stephen A (2017) Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles. Nucleic Acids Res 45:7031-7041
Cannistraro, Vincent J; Pondugula, Santhi; Song, Qian et al. (2015) Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo. J Biol Chem 290:26597-609
Taylor, John-Stephen (2015) Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage. DNA Repair (Amst) 36:59-67
Smith, Jillian E; Lu, Chen; Taylor, John-Stephen (2014) Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences. Nucleic Acids Res 42:5007-19
Song, Qian; Cannistraro, Vincent J; Taylor, John-Stephen (2014) Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle. Nucleic Acids Res 42:13122-33
Taggart, David J; Camerlengo, Terry L; Harrison, Jason K et al. (2013) A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis. Nucleic Acids Res 41:e96
Song, Qian; Sherrer, Shanen M; Suo, Zucai et al. (2012) Preparation of site-specific T=mCG cis-syn cyclobutane dimer-containing template and its error-free bypass by yeast and human polymerase ?. J Biol Chem 287:8021-8
Song, Qian; Cannistraro, Vincent J; Taylor, John-Stephen (2011) Rotational position of a 5-methylcytosine-containing cyclobutane pyrimidine dimer in a nucleosome greatly affects its deamination rate. J Biol Chem 286:6329-35

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