Abstract

Numerous chemical agents react with DNA to form exocyclic modified DNA bases which promote replication errors and mutations, if unrepaired. The long-term goal of this project focuses on identifying human enzymes that remove cyclic adducts from DNA and defining their mechanism of action. The experimental approach utilizes a defined site-specific adduct-containing DNA substrate to probe for and purify DNA-glycosylases from human cell-free extracts. Structurally related adducts incorporated into defined oligonucleotides include 1,N6-etheno A (EA), 3-N4-EC, 1,N2-EG, N2,3-EG, 1,N6-benzetheno A (pBQ-A), 3, N4-pBQ-C, 1,N2, pBQ-G and 1,N6-ethano-A. These adducts are formed after DNA treatment with chloroacetaldehyde and p-benzoquinone. Using cell-free extracts from HeLa or human placenta, novel glycosylases will be identified following cleavage induction and polyacrylamide gel electrophoresis or HPLC detection of the modified exocyclic base. DNA glycosylases that act on these substrates will be purified, characterized, microsequenced and compared in the protein data base to other proteins. The objective is to extend our understanding of enzyme-substrate recognition.
Four specific aims are listed that direct this project. 1) To construct site-specific oligonucleotides containing various nucleotides modified by bifunctional agents. The detailed synthetic chemistry, phosphoramidite, and oligoneucleotide synthesis will be conducted under other NIH grants (CA47723 and ES07363). 2) To test for excision of these modified bases in human cell-free extracts. Band-shift, binding kinetics and footprinting experiment will be conducted to describe the enzyme-substrate interaction. 3) To investigate the range of substrate specificity and sequence context effects of repair rates for EC-DNA glycosylase, benzethenoA-DNA glycosylase and benzetheno-C-DNA glycosylase. 4) To purify to homogeneity recently identified or other new human glycosylase, characterize the activities and obtain protein micro-sequencing information.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA072079-04
Application #
2911360
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1996-09-05
Project End
2002-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Lawrence Berkeley National Laboratory
Department
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720

  Publications

Rodriguez, Ben; Yang, Yanu; Guliaev, Anton B et al. (2010) Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA. Toxicol Lett 193:26-32
Chenna, Ahmed; Gupta, Ramesh C; Bonala, Radha R et al. (2008) Synthesis of the fully protected phosphoramidite of the benzene-DNA adduct, N2-(4-Hydroxyphenyl)-2'-deoxyguanosine and incorporation of the later into DNA oligomers. Nucleosides Nucleotides Nucleic Acids 27:979-91
Hang, Bo; Guliaev, Anton B (2007) Substrate specificity of human thymine-DNA glycosylase on exocyclic cytosine adducts. Chem Biol Interact 165:230-8
Wang, Ping; Guliaev, Anton B; Elder, Rhoderick H et al. (2006) Alkylpurine-DNA-N-glycosylase excision of 7-(hydroxymethyl)-1,N6-ethenoadenine, a glycidaldehyde-derived DNA adduct. DNA Repair (Amst) 5:23-31
Wang, Ping; Guliaev, Anton B; Hang, Bo (2006) Metal inhibition of human N-methylpurine-DNA glycosylase activity in base excision repair. Toxicol Lett 166:237-47
Xie, Zhongwen; Zhang, Yangbin; Guliaev, Anton B et al. (2005) The p-benzoquinone DNA adducts derived from benzene are highly mutagenic. DNA Repair (Amst) 4:1399-409
Guliaev, Anton B; Singer, B; Hang, Bo (2004) Chloroethylnitrosourea-derived ethano cytosine and adenine adducts are substrates for Escherichia coli glycosylases excising analogous etheno adducts. DNA Repair (Amst) 3:1311-21
Guliaev, Anton B; Hang, Bo; Singer, B (2004) Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C. Nucleic Acids Res 32:2844-52
Hang, Bo (2004) Repair of exocyclic DNA adducts: rings of complexity. Bioessays 26:1195-208
Hang, Bo; Singer, B (2003) Protein-protein interactions involving DNA glycosylases. Chem Res Toxicol 16:1181-95

Showing the most recent 10 out of 26 publications