Tobacco-smoking is the single major cause of cancer mortality in the US, and is a risk factor for a number of cancers including lung, upper aero-digestive tract, bladder and pancreas. One of the most powerful carcinogens in tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK is bioactivated to potent electrophiles that react to form methyl and 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts. Role of methyl-DNA adducts in carcinogenesis have been well characterized and the current paradigm is that methyl- DNA adducts are more important than POB-DNA adducts in the etiology of tobacco-induced cancers. However, recently it was found that O2-POB-dT adduct is the most persistent POB adduct in NNK-treated rodents. Our preliminary results show that O2-POB-dT is inefficiently repaired in human cells and is mutagenic in SOS-induced E. coli and mammalian cells. The objective of this application is to determine the mechanisms by which O2-POB-dT forms mutations and is repaired in mammalian cells. These goals will be examined in two specific aims: (1) to determine the polymerases involved in accurate and mutagenic bypass of O2-POB-dT and (2) to determine the mechanisms by which the POB-adducts are repaired. The polymerases involved in the bypass will be determined in cells in which specific polymerases are down regulated by siRNA, and in vitro with purified polymerases. The role of strand switching during translesion synthesis will be examined with cell- free extracts. The repair mechanisms will be evaluated by a combination of ex vivo and in vitro experiments. The roles of NER and BER will be evaluated ex vivo using cells with deficient repair proteins using a HPLC- MS/MS assay to measure levels of DNA adducts. The repair of O2-POB-dT via NER and BER will examined in vitro using synthetic oligodeoxynucleotides. The role of transcription-coupled NER for O6-POB-dG and O2- POB-dT will be probed with a novel modified host cell reactivation.

Public Health Relevance

One way by which tobacco smoke causes cancer is by damaging the DNA in the cell. If the damage is not repaired then when the cell replicates its DNA mistakes (mutations) are made that can lead to cancer. The goal of this project is to understand how the cell repairs and replicates an important class of DNA damage.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES021762-02
Application #
8514612
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Shaughnessy, Daniel
Project Start
2012-07-20
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
2
Fiscal Year
2013
Total Cost
$332,641
Indirect Cost
$51,721
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Gowda, A S Prakasha; Spratt, Thomas E (2016) DNA Polymerases η and ζ Combine to Bypass O(2)-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens. Chem Res Toxicol 29:303-16
Patra, Amritraj; Politica, Dustin A; Chatterjee, Arindom et al. (2016) Mechanism of Error-Free Bypass of the Environmental Carcinogen N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase η. Chembiochem 17:2033-2037
Bose, Arindam; Pande, Paritosh; Jasti, Vijay P et al. (2015) DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8-2'-deoxyguanosine adduct of the dietary mutagen IQ in human cells. Nucleic Acids Res 43:8340-51
Gowda, A S Prakasha; Moldovan, George-Lucian; Spratt, Thomas E (2015) Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency. J Biol Chem 290:16292-303
Pattammattel, Ajith; Williams, Christina L; Pande, Paritosh et al. (2015) Biological relevance of oxidative debris present in as-prepared graphene oxide. RSC Adv 5:59364-59372
Weerasooriya, Savithri; Jasti, Vijay P; Bose, Arindam et al. (2015) Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells. DNA Repair (Amst) 35:63-70
Pande, Paritosh; Haraguchi, Kazuhiro; Jiang, Yu-Lin et al. (2015) Unlike catalyzing error-free bypass of 8-oxodGuo, DNA polymerase λ is responsible for a significant part of Fapy·dG-induced G → T mutations in human cells. Biochemistry 54:1859-62
Politica, Dustin A; Malik, Chanchal K; Basu, Ashis K et al. (2015) Base-Displaced Intercalated Structure of the N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone DNA Adduct. Chem Res Toxicol 28:2253-66
Pande, Paritosh; Malik, Chanchal K; Bose, Arindam et al. (2014) Mutational analysis of the C8-guanine adduct of the environmental carcinogen 3-nitrobenzanthrone in human cells: critical roles of DNA polymerases η and κ and Rev1 in error-prone translesion synthesis. Biochemistry 53:5323-31
Pednekar, Varsha; Weerasooriya, Savithri; Jasti, Vijay P et al. (2014) Mutagenicity and genotoxicity of (5'S)-8,5'-cyclo-2'-deoxyadenosine in Escherichia coli and replication of (5'S)-8,5'-cyclopurine-2'-deoxynucleosides in vitro by DNA polymerase IV, exo-free Klenow fragment, and Dpo4. Chem Res Toxicol 27:200-10

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