Education:

B.Sc., Moscow State University, Russia, 1988
M.S., Moscow State University, Russia, 1990
Ph.D., University of North Carolina at Chapel Hill, 1997
Postdoctoral, Massachusetts Institute of Technology, 1999

Course Links:

Phar 6151 - Biochemistry of Medicinals I
Phar 6152 - Biochemistry of Medicinals II

Research Interests:

Humans are exposed to complex mixtures of environmental and dietary carcinogens, as well as endogenous electrophiles produced from normal metabolism. Some of these chemicals and their metabolites are electrophilic species capable of chemical reactions with DNA. The resulting covalently modified DNA nucleobases (DNA adducts) have distorted base pairing characteristics and, if not repaired, can be converted to heritable mutations during DNA replication. Carcinogen-induced changes in critical genes that control cell growth and differentiation can lead to the initiation of cancer. Paradoxically, many common antitumor drugs use a similar mechanism by alkylating DNA in an attempt to induce tumor cell death. Long-term cancer survivors are at risk for secondary cancers resulting from exposure to antineoplastic agents. The focus of our research is to investigate the structural basis for promutagenic and anticancer activity of DNA-modifying agents. The primary experimental tool employed in our laboratory is biological mass spectrometry (ESI-MS, cap HPLC-MS, MS/MS, and MALDI-TOF MS), although most of the projects also involve organic synthesis, structural analyses (e.g. NMR, 2D NMR, CD spectroscopy), and molecular modeling of chemically altered DNA.

DNA damage by tobacco carcinogens and lung cancer
Smoking is responsible for at least 85% of total lung cancer cases in the US. Tobacco smoke contains over 60 known lung carcinogens, including tobacco-specific nitrosamines and polycyclic aromatic hydrocarbons. DNA damage induced by carcinogens present in cigarette smoke (tobacco-specific nitrosamines and polycyclic aromatic hydrocarbons) is investigated in order to identify the causative agents for smoking-related lung cancer. Our hypothesis is that the distribution of lesions in DNA treated with the chemical agents involved in cancer initiation parallels the mutational spectra observed in tumors. We have developed a mass spectrometry-based approach to map the locations of carcinogen-induced DNA damage within critical genes. In this approach, stable isotope labeled nucleobases are inserted at specific position within synthetic DNA duplexes, and the extent of reaction at the labeled base is determined by mass spectrometry. The ultimate goal of this research is to elucidate the biological mechanisms of chemical carcinogens by establishing a link between specific types of DNA damage and the genetic changes observed in human cancers.

DNA-DNA and DNA-protein cross-linking agents as carcinogens and drugs
Bifunctional antitumor drugs and carcinogens, e.g. nitrogen mustards and 1,2,3,4-diepoxybutane, are an interesting group of biologically active compounds. These molecules are capable of sequentially reacting with two sites within the DNA duplex, giving rise to DNA cross-links. Depending on their structure, DNA cross-links can either induced cell death (this is the basis of action of bifunctional antitumor drugs) or give rise to heritable mutations. It is important to identify the structural features of the cross-linked lesions that render them biologically active. Our laboratory is investigating the structural identities and biochemical characteristics of DNA cross-links induced by antitumor nitrogen mustards and carcinogenic 1,2,3,4-diepoxybutane in an attempt to determine the structural basis for their differing biological effects. Molecular dynamics simulations are employed to analyze the impact of DNA-DNA cross-links on local DNA structure.

Cross-linking of DNA-binding proteins to their DNA targets
In addition to DNA-DNA cross-links, bifunctional lesions involving DNA and proteins are likely to play an important role in cytotoxicity of bifunctional electrophiles. DNA binding proteins such histones, High Mobility Group (HMG) proteins, transcription factors, and DNA repair proteins, are at risk of becoming covalently linked to their DNA targets in the presence of bifunctional alkylating agents. The resulting bulky adducts block DNA replication and transcription. Importantly, many DNA repair proteins are overproduced in tumor cells. We hypothesize that the cross-linking of DNA repair proteins to their DNA targets contributes to the mechanisms by which tumor cells are sensitized to anticancer DNA-damaging drugs.

In collaboration with Prof. Anthony Pegg at Penn State, we are investigating the cross-linking of human O⁶-alkylguanine DNA alkyltransferase (AGT) to DNA in the presence of 1,2,3,4-diepoxybutane (DEB). Initial DNA reactions with DEB generate N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine monoadducts which retain one of the epoxy groups. We have shown that AGT binding to DEB-modified DNA leads to the formation of DNA-protein lesions that involve the active site cysteine of AGT (Cys 145). This was determined by HPLC-ESI-MS/MS analyses of tryptic digests of AGT and peptide sequencing. Studies are now in progress at our laboratory involving other DNA-binding proteins (e.g. histine H4) and bifunctional electrophiles (nitrogen mustards) to establish the role of DNA-protein cross-linking in their biological activity.

DNA oxidation by endogenous reactive oxygen species
The oxidative degradation of DNA has been implicated in aging, cancer, and many degenerative diseases. Because of their lowest redox potential, guanine bases in DNA are frequently targeted for oxidation. Multiple oxidation products are formed, including 8-oxoguanine (8-oxo-G) and 2,2-diamino-4-[(2-deoxy-b-D-erythro-pentofuranosyl)amino]-2,5-dihydrooxazol-5-one (oxazolone). We are investigating the mechanisms of guanine oxidation in the presence of reactive oxygen species. Highly sensitive and specific detection of oxidized DNA bases by capillary HPLC- electrospray mass spectrometry is used to quantify oxidative DNA damage in breast cancer and diabetes. The final goal of this research is to identify new biomarkers of oxidative stress that can potentially serve as indicators of carcinogenic risk.

More detailed information about our research group is available.

Recent Publications:

Park, S., Hodge, J., Anderson, C., and Tretyakova, N. (2004) Guanine-adenine DNA cross-linksing by 1,2,3,4-diepoxybutane: potential basis for biological activity. Chemical Research in Toxicology, 17, 1638-1651.

Rajesh, M., Wang, G., Jones, R., and Tretyakova, N.Y. (2005) Mapping NNK adducts in p53 gene derived DNA sequences by stable isotope labeling. Biochemistry, 44, 2197-2207.

Park, S., Anderson, C., Loeber, R., Seetharaman, M., Jones, Roger, Tretyakova, N. Interstrand and Intrand DNA-DNA Cross-Linking by 1,2,3,4-Diepoxybutane; Role of Sterochemistry.  Journal of the American Chemical Society. 127(41) 14355-14365.

Kim, J., Park, S., Tretyakova, N., Wagner, C. R. (2005)  A Method for Quantitating the Intracelluar Metabolism of AZT Amino Acid Phosphoramidate Pronucleotides by Capillary High-Performance Liquid Chromatography-Electrospray lonization Mass Spectrometry.  Molecular Pharmaceutics.  2(3) 233-241.

Guza, R., Rajesh, M., Pegg, and Natalia Tretyakova. (2006)  Kinetics of O⁶-Me-dG repair by O⁶- Alkylguanine DNA Alkyltransferase within K-ras Gene-Derived DNA Sequences. Chemical Research in Toxicology, 19(4) 531-538.

Matter, B., Malejka-Giganti, D., Csallany, A.S., and Tretyakova, N. (2006) Quantitative analysis of the oxidative DNA lesions, 2,2-diamino-4- [(2-deoxy-(-D-erithro-pentofuranosyl) amino] -2,5-dihydrooxazol-5-one in vitro and in vivo by isotope dilution. Nucleic Acids Research. 34, 5449-5460.

Loeber, R., Rajesh, M., Pegg, A., and Tretyakova, N. (2006) Cross-linking of the human DNA repair protein O⁶-alkylguanine DNA alkyltransferase to its DNA substrate in the presence of 1,2,3,4-diepoxybutane.Chemical Research in Toxicology,19 (5) (Cover Article), 645-654.

Malejka-Gigant, D. Tretyakova, N. (2006)  Molecular Mechanisms of Carcinogenesis.  Carcinogenic and Anticarcinogenic Food Components. 13-36.

Tretyakova, N.  Livshits, A.,  Park, S., Bisht, B., and Goggin, M.  (2007) Structural elucidation of a novel DNA-DNA cross-link of 1,2,3,4-diepoxybutane. Chemical Research in Toxicology 20(2);284-289

Goggin, M., Loeber, R., Park, S., Walker, V., and Tretyakova, N. (2007) Quantitative HPLC-ESI-MS/MS method for bis-N7-G-butanediol adducts of 1,2,3,4-diepoxybutane. Chemical Research in Toxicology, 20 (5), 839-847

Antsypovich, S., Quirk Dorr, D., Pitts, C., and Tretyakova, N. (2007) Site Specific   N⁶ -(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine Oligodeoxynucleotide Adducts of 1,2,3,4-Diepoxybutane:Synthesis and Stability at Physiological pH.Chemical Research in Toxicology 20 (4), 641-649

Miksa, Chinnappan, R., Dang, N., Reppert, M., Matter, B., Tretyakova, N., Grubor, N., and Jankowiak, R. (2007) Spectral Differentiation and Immunoaffinity Capillary Electrophoresis Separation of Enantiomeric Benzo(a)pyrene Diol Epoxide-Derived DNA Adducts, Chemical Research in Toxicology, 20(8), 1192-1199

Matter, B., Guza, R., Zhao, J., Li, Z., Jones, R., and Tretyakova, N. (2007) Sequence Distribution of Acetaldehyde-Derived N^2-Ethyl-dG Adducts along Duplex DNA Chemical Research in Toxicology ASAP articles

Loeber, R., Quirk Dorr, D., Rajesh, M., and Tretyakova, N.  DNA-protein cross-linking by antitumor nitrogen mustards. Submitted

Quirk Dorr, D., Pitts, C., and Tretyakova, N. Synthesis of DNA oligodeoxynucleotides containing  N⁶ -(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine adducts of 1,2,3,4-diepoxybutane. Chemical Research in Toxicology:In Press.