EXCEED THE SPACE PROVIDED. This projectfocuses on the distributionof specifictobacco carcinogen-DNA adducts within the two genes frequently targeted in smoking induced lun9 cancer: the p53 tumor suppressor gene and the K-ras proto-oncogene. Lung tumors of smokersoften contain characteristic'hotspots' for point mutationswithin K-ras codon12 and p53 codons 157,158, 248, and 273. Thesemutations are believed to be a result of DNA polymerase errors durin9 the replication of DNA chemicallymodified by metabolically activated tobacco carcinogens. The large numbers of mutations atspecific sites of the K-rasand p53 genes may originatefrom their high reactivity towards tobacco carcinogens, deficientrepair of DNA lesions,or elevatedmispairin9 rates for the lesions within specificsequencecontext. While the mispairing rates of DNAlesions introducedin a defined sequenceenvironmentcan be accuratelyestablishedby sitespecific mutagenesis,analytical methods capableof mapping the formationand repair of specific DNA lesions within gene sequencesare lacking.The presentstudy will use a mass spectrometry.basedapproach recently developedin this laboratoryto quantifythe formationof DNA adducts at specific positionswithin p53 and K-ras.derived DNA sequences. The research willfocus on DNA damage induced by two prominenttobacco carcinogens, benzo[a]pyrene(B[a]P) and 4.(methylnitrosamino)-l-(3.pyridyl)-l-butanone (NNK). Double.stranded,synthetic oligodeoxynucleotidesrepresenting mutation-prone regions of the p53 and K-ras genes will be treated with reactive metabolitesof B[a]Pand NNK, and the extent of adduct formationat each positionwill be determinedby liquidchromatography.electrospray ionizationtandem mass spectrometryin combination with stableisotope labeling. The same approachwill be used to analyzethe effect of endogenouscytosine methylation on the formationof tobaccocarcinogen-inducedguanine lesionsand to determinethe effect of K-ras and p53 sequencecontext on the repair of NNK-inducedO6.alkylguanine adducts.These studieswill 1) Establish the distributionof B[a]Pdiolopoxide.induced nucleobaselesions within K-ras and p53-derived DNA sequencescontaining knownmutational hotspots. 2) Mapthe formation of NNK.inducedmothylated and pyridyloxobutylatedlesions along K-ras and p53-derivedDNA sequences. 3) Analyzethe effectsof endogenouscytosine methylationon the formation of B[a]P and NNK adducts at neighboringguanines. 4) Examinethe effects of K-ras sequencecontexton O6.alkylguanine DNA alkyltransferase-catalyzedrepair of NNK.inducedO6.alkylguanine lesions.The resultsof this work will afford new insights into the molecularbasis of genetic changesobserved in smoking-inducedlungcancer and will aid in the developmentof rationalpreventionstrategiesand mechanism-basedbiomarkersfor individuals at risk. This research will also laya foundationfor future in vivo studiesof carcinogen-modifiednucleobasesat singlenucleotide resolution. PERFORMANCE SITE ========================================Section End===========================================
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