Abstract

The broad long-term goal of this project is to understand how mutations occur from the replication of carcinogen-modified DNA. The objective of this grant proposal is to elucidate the interactions between DNA polymerase and the minor groove of DNA.
The specific aims are to determine which sites in the minor groove of the DNA interact with the DNA polymerase during replication. These interactions will be examined during the replication of the normal DNA bases and theta6-methylguanine, a carcinogen- modified base. Differences observed between correct and incorrect replication will give clues as to the mechanisms that polymerases use to replicate DNA with high fidelity. The health-relatedness of the project is in carcinogenesis. Mutations that occur during replication of normal DNA and carcinogen-modified DNA can lead to cancer. Understanding the mechanism of the formation of mutations might alloW us to predict which compounds are mutagens. The research design is to synthesize DNA analogs in which sites in the minor groove are altered so that they can not participate in hydrogen bonds. The kinetics of incorporation of modified nucleotide triphosphates opposite modified DNA will be measured. Large reduction in rates of replication will indicate that the altered site was involved in a hydrogen bond with the polymerase.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA075074-03
Application #
2896080
Study Section
Special Emphasis Panel (ZRG2-CPA (02))
Program Officer
Okano, Paul
Project Start
1997-07-16
Project End
2000-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Institute for Cancer Prevention
Department
Type
DUNS #
City
Valhalla
State
NY
Country
United States
Zip Code
10595

  Publications

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