This research focusses on the biological consequences of DNA damage with the overall goal of elucidating primary events in carcinogenesis. A principal theme is to establish relationships between the structure of damaged DNA and the functions of enzymes involved in DNA replication and repair. Novel experimental systems have been developed for this purpose. Site specific mutagenesis involves a strategy in which a shuttle plasmid vector, containing a single defined lesion, is allowed to replicate in mammalian cells or bacteria. The position of mutations induced is established by DNA sequence analysis. Primer-extension reactions, catalyzed by DNA polymerase, coupled with steady-state kinetic analysis, are used to explore translesional synthesis and mutagenic events in vitro.
Our specific aims are (a) to establish models for frameshift mutagenesis in terms of misaligned DNA templates and kinetics governing translesional synthesis; (b) to elucidate the molecular basis underlying sequence context effects on base substitutions and deletions; (c) to understand the role of SOS functions in translesional synthesis; (d) to discover pathways by which mutations are generated during repair of bistrand abasic sites in DNA; (e) to demonstrates differences between DNA polymerases in their abilities to generate mutations arising from DNA damage; (f) to develop in vitro assays that predict mutagenic specificity for defined DNA lesions in vivo; (g) to explore mechanisms by which DNA damage enhances the frequency of homologous recombination in mammalian cells and bacteria; and (h) to establish the solution structure of misaligned intermediates formed during deletion mutagenesis. Additional studies are designed (a) to determined the substrate specificity of Fpg protein; (b) to establish the role of the N-terminus in the catalytic function of this enzyme; (c) to reveal the structural basis for binding of the zinc finger domain oxidatively-damaged DNA; (d) to elucidate a catalytic mechanism for DNA glycosylate activity; (e) to detect functional groups on Fpg protein and its substrates that facilitate """"""""recognition"""""""" of oxidative damage; (f) to establish the structure of complexes formed between Fpg protein or adenine DNA glycosylate and analogs of their DNA substrates; and (g) to study substrate binding and mechanism of action of selected AP endonucleases, and (h) to quantify the contribution of Fpg protein to DNA repair in cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA017395-23
Application #
2007200
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1977-01-01
Project End
1998-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
23
Fiscal Year
1997
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Gao, Shujuan; Honey, Sangeet; Futcher, Bruce et al. (2016) The non-homologous end-joining pathway of S. cerevisiae works effectively in G1-phase cells, and religates cognate ends correctly and non-randomly. DNA Repair (Amst) 42:1-10
Kuznetsov, Nikita A; Bergonzo, Christina; Campbell, Arthur J et al. (2015) Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition. Nucleic Acids Res 43:272-81
Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V et al. (2013) DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site. J Biol Chem 288:28936-47
Kuznetsov, Nikita A; Koval, Vladimir V; Zharkov, Dmitry O et al. (2012) Conformational dynamics of the interaction of Escherichia coli endonuclease VIII with DNA substrates. DNA Repair (Amst) 11:884-91
Bergonzo, Christina; Campbell, Arthur J; de los Santos, Carlos et al. (2011) Energetic preference of 8-oxoG eversion pathways in a DNA glycosylase. J Am Chem Soc 133:14504-6
Kirpota, Oleg O; Endutkin, Anton V; Ponomarenko, Michail P et al. (2011) Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase. Nucleic Acids Res 39:4836-50
Mechetin, Grigory V; Zharkov, Dmitry O (2011) Mechanism of translocation of uracil-DNA glycosylase from Escherichia coli between distributed lesions. Biochem Biophys Res Commun 414:425-30
Song, Kun; Campbell, Arthur J; Bergonzo, Christina et al. (2009) An Improved Reaction Coordinate for Nucleic Acid Base Flipping Studies. J Chem Theory Comput 5:3105-13
Song, Kun; Hornak, Viktor; de los Santos, Carlos et al. (2008) Molecular mechanics parameters for the FapydG DNA lesion. J Comput Chem 29:17-23
Song, Kun; Kelso, Catherine; de los Santos, Carlos et al. (2007) Molecular simulations reveal a common binding mode for glycosylase binding of oxidatively damaged DNA lesions. J Am Chem Soc 129:14536-7

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