The proposed studies address the basis for an unusual DNA sequence selectivity for damage produced by nitrosoperoxycarbonate (ONOOCO2-), a chemical mediator of inflammation. In a manner opposite to """"""""classical"""""""" one electron DNA oxidants, the reactivity of ONOOCO2- toward guanine (G) in DNA was observed to increase as a function of the calculated sequence-dependent G ionization potential. In contrast, G reactivity toward photooxidation decreases with increasing ionization potential. We propose to investigate this phenomenon to test several hypotheses: (1) that the behavior of ONOOCO2- is common to negatively-charged oxidants; (2) that the behavior reflects preferential oxidation of G's that are more solvent exposed; and (3) that the initial one-electron oxidation event determines the selectivity of G oxidation by ONOOCO2- and other oxidants. The results will provide new insights into the mechanistic link between inflammation and cancer by revealing the chemical basis for the location of DNA damage and subsequent mutations.
Aim 1 : Define the DNA sequence selectivity of carbonate radical anion (CO3.), ONOO-, ONOOCO2- and related oxidants. We will extend preliminary studies to include a range of DNA sequences (oligos, 32P-DNA fragments and genomic DNA) and ONOOCO2- concentrations. We will also test the hypothesis that the reactivity of a G in DNA is influenced by the charge of the oxidant by comparing the sequence selectivity of ONOOCO2- to that for a variety of negatively-charged, neutral and positively-charged oxidants.
Aim 2 : Define the basis for the sequence selectivity of CO3., ONOO- and ONOOCO2-. First, we test the hypothesis that the sequence selectivity of G oxidation arises at the first step of the oxidation reaction and not as a result of later product formation. We then proceed to test the hypothesis that solvent exposure governs the reactivity of G's toward ONOOCO2- and other oxidants by: (1) comparing sequence selectivity in single- and double-stranded oligos; and (2) assessing the imino proton exchange rates of G's in different DNA sequence contexts.
Aim 3 : Compare the sequence selectivity of DNA damage chemistry for CO3. and ONOOCO2-. To complement Aims #1 and #2, we will test the hypothesis that the products of G oxidation vary as a function of sequence context. First, we define the proportions of base and deoxyribose oxidation in plasmid DNA treated with the oxidants and then proceed to investigate the role of DNA sequence context in the spectrum of G oxidation products. Guanine lesions in the oligos will be characterized by LC-MS for single G sequences and by exonuclease digestion/MALDI-MS for localizing damage in sequences with multiple G's.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA110261-05
Application #
7393749
Study Section
Special Emphasis Panel (ZRG1-ONC-L (02))
Program Officer
Okano, Paul
Project Start
2004-07-01
Project End
2010-04-30
Budget Start
2008-05-01
Budget End
2010-04-30
Support Year
5
Fiscal Year
2008
Total Cost
$309,783
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Organized Research Units
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Hao, Yi-Heng; Doyle, Jennifer M; Ramanathan, Saumya et al. (2013) Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination. Cell 152:1051-64
Lim, Kok Seong; Cui, Liang; Taghizadeh, Koli et al. (2012) In situ analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine oxidation reveals sequence- and agent-specific damage spectra. J Am Chem Soc 134:18053-64
Lim, Kok Seong; Taghizadeh, Koli; Wishnok, John S et al. (2012) Sequence-dependent variation in the reactivity of 8-Oxo-7,8-dihydro-2'-deoxyguanosine toward oxidation. Chem Res Toxicol 25:366-73
Lonkar, Pallavi; Dedon, Peter C (2011) Reactive species and DNA damage in chronic inflammation: reconciling chemical mechanisms and biological fates. Int J Cancer 128:1999-2009
Yun, Byeong Hwa; Dedon, Peter C; Geacintov, Nicholas E et al. (2010) One-electron oxidation of a pyrenyl photosensitizer covalently attached to DNA and competition between its further oxidation and DNA hole injection. Photochem Photobiol 86:563-70
Margolin, Yelena; Dedon, Peter C (2010) A general method for quantifying sequence effects on nucleobase oxidation in DNA. Methods Mol Biol 610:325-40
Barth, Marita; Dederich, Debra; Dedon, Peter (2009) An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA. Biotechniques 47:633-5
Dedon, Peter C (2008) The chemical toxicology of 2-deoxyribose oxidation in DNA. Chem Res Toxicol 21:206-19
Margolin, Yelena; Shafirovich, Vladimir; Geacintov, Nicholas E et al. (2008) DNA sequence context as a determinant of the quantity and chemistry of guanine oxidation produced by hydroxyl radicals and one-electron oxidants. J Biol Chem 283:35569-78
Taghizadeh, Koli; McFaline, Jose L; Pang, Bo et al. (2008) Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry. Nat Protoc 3:1287-98

Showing the most recent 10 out of 15 publications