The long-term goal of this project is to understand the chemical interactions that govern replication of DNA. The short-term object of this research proposal is to determine the role that hydrogen bonding, between the polymerase and the minor groove of the DNA, plays in the fidelity of DNA replication. The critical interactions that control the fidelity of DNA replication occur in the transition states of several of the individual steps that comprise DNA synthesis. The transition states will be probed by structure activity relationships. The structure of the reactants will be altered by atomic substitution of the DNA and amino acid substitution of the polymerases, and the progress of the reaction will be measured with pre-steady-state kinetics. Two polymerases will be studied, DNA polymerase I of E. coli and mammalian DNA polymerase beta. The biochemical mechanisms of DNA synthesis by these proteins have been well studied, thus providing a firm foundation for structure-function analyses. Moreover, as the enzymes display structural differences, they may utilize different mechanisms for fidelity control. The following specific hypotheses will be tested. (1) Hydrogen bonds between polymerase and the minor groove of DNA are crucial to catalysis and fidelity of DNA replication. In particular, Arg283 of polymerase beta makes a crucial hydrogen bond to the N3-position of a purine of the template base. (2) Arg668 of E. coli DNA polymerase I acts as a sensor for the correct geometry at the terminal base pair through interactions with N3 of guanine and ring oxygen of the incoming dNTP. (3) The interactions between polymerases and the minor groove of DNA are dependent on the sequence of the DNA. (4) The interactions between polymerases and the O2-positions of dTTP and dCTP are important due to steric interactions between the nucleotide and the protein. (5) The low fidelity bypass polymerases use minor groove interactions to replicate DNA. The results of these experiments will provide very specific mechanistic information of how polymerases replicate DNA with high fidelity. This knowledge will add to our fundamental knowledge of DNA replication.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA075074-05
Application #
6621039
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1997-07-16
Project End
2005-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
5
Fiscal Year
2003
Total Cost
$261,013
Indirect Cost
Name
Institute for Cancer Prevention
Department
Type
DUNS #
City
Valhalla
State
NY
Country
United States
Zip Code
10595
Chen, Gang; Dellinger, Ryan W; Sun, Dongxiao et al. (2008) Glucuronidation of tobacco-specific nitrosamines by UGT2B10. Drug Metab Dispos 36:824-30
DeCarlo, Lindsey; Gowda, A S Prakasha; Suo, Zucai et al. (2008) Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV. Biochemistry 47:8157-64
Coulter, Richard; Blandino, Maureen; Tomlinson, Jessica M et al. (2007) Differences in the rate of repair of O6-alkylguanines in different sequence contexts by O6-alkylguanine-DNA alkyltransferase. Chem Res Toxicol 20:1966-71
Kretulskie, Angie M; Spratt, Thomas E (2006) Structure of purine-purine mispairs during misincorporation and extension by Escherichia coli DNA polymerase I. Biochemistry 45:3740-6
McCain, Melodie D; Meyer, Aviva S; Schultz, Sherri S et al. (2005) Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli. Biochemistry 44:5647-59
Meyer, Aviva S; Blandino, Maureen; Spratt, Thomas E (2004) Escherichia coli DNA polymerase I (Klenow fragment) uses a hydrogen-bonding fork from Arg668 to the primer terminus and incoming deoxynucleotide triphosphate to catalyze DNA replication. J Biol Chem 279:33043-6
Meyer, Aviva S; McCain, Melodie D; Fang, Qingming et al. (2003) O6-alkylguanine-DNA alkyltransferases repair O6-methylguanine in DNA with Michaelis-Menten-like kinetics. Chem Res Toxicol 16:1405-9
Washington, M Todd; Wolfle, William T; Spratt, Thomas E et al. (2003) Yeast DNA polymerase eta makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate. Proc Natl Acad Sci U S A 100:5113-8
Spratt, T E (2001) Identification of hydrogen bonds between Escherichia coli DNA polymerase I (Klenow fragment) and the minor groove of DNA by amino acid substitution of the polymerase and atomic substitution of the DNA. Biochemistry 40:2647-52
Spratt, T E; Wu, J D; Levy, D E et al. (1999) Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase. Biochemistry 38:6801-6