Ph.D. 2001 (Biomolecular Chemistry), New York University, M.S. 1998, New York University , M. S. 1996 (Chemistry; Specialty: Chemistry of Natural Compounds), Lomonosov Moscow State University, Moscow, Russia.

Thesis title: "Influence of Site-Specific Carcinogen DNA adducts on DNA Replication and Transcription"

Research Interest:

Honors:  

Project Description

Mechanistic impact of carcinogen-DNA lesions on DNA replication and mutations.  Understanding the mechanisms by which the presence of these bulky carcinogens can cause DNA replication errors and mutations, is a key goal in the field of chemical carcinogenesis.  While numerous studies have addressed this important problem, this goal remains an elusive objective. The focus of my work is the fundamental and systematic investigation of the role of the different biochemical factors that impact on these phenomena.  We have designed simplified in vitro systems in which the different steps of DNA replication and transcription are analyzed systematically.  Recently, we have shown, for the first time, that the initial step of DNA synthesis, the formation of the primary complex between DNA and a polymerase molecule, is significantly altered by the presence of these bulky carcinogen groups. We have also shown that the chemical rates of  DNA synthesis are markedly affected by the stereochemical properties of the carcinogen (the stereochemistry defines the orientation in space of the different chemical groups).   I discovered that stereoisomeric PAH-DNA lesions have markedly different effects on the outcome of polymerase-catalyzed DNA replication.  Furthermore, polymerases extracted from different prokaryotic organisms and mammalian cells, exhibit remarkable differences in the treatment of these pre-mutagenic and pre-carcinogenic PAH-DNA lesions. Our preliminary results have shown that the effects of the mirror-image stereochemistry of the PAH carcinogen-DNA adducts on polymerase catalyzed DNA synthesis are reversed if  polymerases from two different families are used.  This clearly points to differences in the response of different biological organisms to cancer-causing chemicals.   This fascinating observation opens up a new area of studying how genetically related, similar polymerases, deal with aberration in DNA structure caused by  carcinogenic chemicals.

Impact of bulky carcinogen-DNA lesions on transcription.

DNA mutagenesis is not the only way in which the bulky lesions in DNA can cause cancer.  Error-prone transcription of DNAis another pathway. We have succeeded in demonstrating that the presence of bulky carcinogenic PAH groups in highly conserved regulatory DNA transcription sequences can affect the recognition of these sequences by critically important transcription factors. 

The TATA Binding Protein (TBP) is an essential component of the transcription initiation  complex that recognizes and binds  to the minor groove of  the TATA  DNA duplex consensus sequences.  The goal of this project is to determine the effect of a  carcinogen-modified adenine residue on the binding of  TBP,  positioned site-specifically within a regulatory TATA DNA sequence, on the binding of  TBP.  Two key adenine residues within TATA recognition sequence were modified site- and stereospecifically by the bulky carcinogen group. We evaluated formation of TBP complexes with carcinogen-modified TATA DNA by electrophoretic mobility shift assays. The formation of the biologically significant monomeric TBP-DNA complexes was dramatically increased or decreased, depending on the position or stereochemistry of the lesion. The figure on the main lab page represents molecular docking of carcinogen-modified adenine residue into the known TATA box-TBP structure [Nikolov, D. B. et al.  (1996) Proc. Natl. Acad. Sci. USA, 4862-4867]. Native gel electrophoresis experiments with the same duplexes without TBP indicate that none of the modified sequences exhibit unusual bending induced by the lesions, nor that they differ from one another in this respect.  These results suggest that the hydrophobic, bulky BPDE residues influence the binding of TBP by mechanisms other than pre-bending. The efficiency of RNA transcription of TBP-controlled promoters could be strongly influenced by the presence of such bulky lesions that could adversely affect the levels of gene expression.

PUBLICATIONS:  

1.   O. Rechkoblit, J. Krzeminsky, S. Amin, B. Jernström, N. Louneva,  and N. E. Geacintov. Influence of bulky polynuclear carcinogen lesions in a TATA promoter sequence on TATA binding protein-DNA complex formation. (2001) submitted to Biochemistry.

2.   Y.B. Zhang, F.H. Yuan, X.H. Wu, M.Wang, O. Rechkoblit, J.S. Taylor, N.E. Geacintov, Z.G. Wang) Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro. (2000) Nucleic Acids Research 28: (21) 4138-4146.

3.   Y.B. Zhang, F.H. Yuan, X.H. Wu, O. Rechkoblit, J.S. Taylor, N.E. Geacintov, Z.G. Wang Error-prone lesion bypass by human DNA polymerase eta. (2000) Nucleic Acids Research 28: (23) 4717-4724 DEC 1 2000.

4.   H. Tsao, O. Rechkoblit, S. Amin, and N. E. Geacintov. Site-specific DNA polymerase kinetics in the immediate vicinity of bulky benzo[a]pyrene diol epoxide-guanine lesions. (2000) Polycyclic Aromatic Compounds 21: (1-4) 1-10 2000.

5.   O. Rechkoblit, S. Amin, and N. E. Geacintov. Primer-length dependence of binding of DNA polymerase I Klenow fragment  to template-primer complexes containing site-specific bulky lesions (1999) Biochemistry 38 (36), 11834 –11843.

MANUSCRIPTS IN PREPARATION:

1.     O. Rechkoblit, N. Chernichenko, S. Amin, and N. E. Geacintov. Influence of complex Formation, Length of Single-Stranded Overhang, and Adduct Stereochemistry on Klenow fragment polymerase-catalazed bypass of [BP]-N²-dG lesions in vitro. Will be submitted to Biochemistry.

2.     O. Rechkoblit, N. Chernichenko, S. Amin, and N. E. Geacintov. Translesional synthesis catalyzed by repair and replicative polymerase: Influence of length of single-stranded overhang and adduct stereochemistry. Will be submitted to Biochemistry.