Many alkylating agents react with DNA to afford predominanfiy N7-adducts of deoxyguanosine (dG). The genotoxicity and cytotoxicity of agents that react primarily at N7-dG is often attributed to other less abundant DNA lesions. The overarching hypothesis of this PPG application is the cationic N7-dG adducts are converted into N5-substituted formamidopyrimidine (Fapy-dG) lesions in cells, and the latter lesions contribute significantly to the genotoxicity and cytotoxicity of alkylating agents. This Program Project interactively uses organic synthesis, bioanalytical chemistry, structural biology, enzymology, and molecular biology to elucidate the molecular details by which Fapy-dG adducts derived from endogenous and exogenous electrophiles and chemotherapeutic agents alter DNA replication and repair. Project 1 will utilize bioanalytical mass spectrometry, chemical synthesis, and enzymology to address this hypothesis. Efforts will focus on formamidopyrimidine (Fapy-dG) lesions derived from methylating agents such as temozolomide (MeFapy-dG), chlorooxirane (OxEt-Fapy-dG), thioTEPA (AE-Fapy-dG) and nitrogen mustards (NM-Fapy-dG), including interstrand cross-links (FapyG-NM-FapyG, and FapyG-NMG). Temozolomide, thioTEPA, and nitrogen mustards are clinically used chemotherapeutic agents and we hypothesize that the corresponding N5-substituted Fapy-dG lesion plays an important role in their mechanism of action.
The Specific Aims of Project 1 will establish the presence of N5-substituted Fapy-dG lesions in mammalian cells (Specific Aim 1), chemically synthesized oligonucleotides containing the desired Fapy-dG lesion in a site-specifically manner (Specific Aim 2), and determine the miscoding potential of the Fapy-dG lesion in vitro (Specific Aim 3). Our ability to site-specifically incorporate the Fapy-dG lesions into oligonucleotide is central to the research plans of Projects 1, 2 and 3. Project 1 will work closely with the DNA Synthesis Resource Core.

Public Health Relevance

Fapy lesions have been an understudied class of DNA adduct largely due to their complex chemistry. We propose to develop methods to understand the chemistry of these lesions, which will aid in their detection, preparation, and further study. The result will provide a better understanding of the role of DNA alkylating agents in human cancers and well as the mechanism of action of chemotherapuetic agents.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA160032-22
Application #
8559506
Study Section
Special Emphasis Panel (ZCA1-RPRB-O)
Project Start
Project End
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
22
Fiscal Year
2013
Total Cost
$1,006,869
Indirect Cost
$224,768
Name
Vanderbilt University Medical Center
Department
Type
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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
O'Flaherty, D K; Patra, A; Su, Y et al. (2016) Lesion Orientation of O(4)-Alkylthymidine Influences Replication by Human DNA Polymerase η. Chem Sci 7:4896-4904
Choi, Jeong-Yun; Patra, Amritaj; Yeom, Mina et al. (2016) Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι. J Biol Chem 291:21063-21073
Egli, Martin (2016) Diffraction Techniques in Structural Biology. Curr Protoc Nucleic Acid Chem 65:7.13.1-7.13.41
Minko, Irina G; Jacobs, Aaron C; de Leon, Arnie R et al. (2016) Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites. Sci Rep 6:28894
Patra, Amritraj; Su, Yan; Zhang, Qianqian et al. (2016) Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase η. J Biol Chem 291:14134-45
Su, Yan; Egli, Martin; Guengerich, F Peter (2016) Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η. J Biol Chem 291:3747-56
Thiaville, Jennifer J; Kellner, Stefanie M; Yuan, Yifeng et al. (2016) Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proc Natl Acad Sci U S A 113:E1452-9
Patra, Amitraj; Zhang, Qianqian; Guengerich, F Peter et al. (2016) Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase η. J Biol Chem 291:24304-24313
O'Flaherty, Derek K; Guengerich, F Peter; Egli, Martin et al. (2015) Backbone Flexibility Influences Nucleotide Incorporation by Human Translesion DNA Polymerase η opposite Intrastrand Cross-Linked DNA. Biochemistry 54:7449-56

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