This proposal focuses on developing an in vitro model to study how DNA replication and repair enzymes act on defined site-directed lesions in DNA. Little is known concerning the repertoire of DNA metabolizing enzymes available in neurons and astrocytes, and how these enzymes respond to DNA damage and vary with age. Two extremely sensitive assays, based on polyacrylamide gel electrophoresis, will be used to study the behavior of DNA synthetic and repair enzymes with three classes of DNA lesions, an abasic (apurinic/apyrimidinic) lesion, alkylated guanine and thymine nucleotide analogues, and 8-oh guanine, a lesion resulting from oxidative damage. The first assay will be used to identify and characterize replication and repair DNA polymerases, using extracts from liver and brian in young and old animals, and in dividing and nondividing cells grown in culture. There is a paucity of data on the ability of the various DNA polymerases to copy and bypass base and sugar modified DNA lesions. The second assay will be used to identify enzymes, such as glycosylases, methyltransferases, endo- and exonucleases, and ligases, involved in lesion repair. We intend to assay crude extracts of brain and liver cells for activities that are induced in response to DNA damage, and to purify and characterize these enzymes. We will determine if animal cells exhibit an induced """"""""error-prone"""""""" response to DNA damage analogous to the SOS response in Escherichia coli and other enterobacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG011398-05
Application #
2052590
Study Section
Neuroscience, Behavior and Sociology of Aging Review Committee (NBSA)
Program Officer
Finkelstein, David B
Project Start
1992-08-01
Project End
1999-04-30
Budget Start
1996-06-01
Budget End
1999-04-30
Support Year
5
Fiscal Year
1996
Total Cost
Indirect Cost
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Hartenstine, M J; Goodman, M F; Petruska, J (2000) Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase. J Biol Chem 275:18382-90
Petruska, J; Hartenstine, M J; Goodman, M F (1998) Analysis of strand slippage in DNA polymerase expansions of CAG/CTG triplet repeats associated with neurodegenerative disease. J Biol Chem 273:5204-10
Bhui-Kaur, A; Goodman, M F; Tower, J (1998) DNA mismatch repair catalyzed by extracts of mitotic, postmitotic, and senescent Drosophila tissues and involvement of mei-9 gene function for full activity. Mol Cell Biol 18:1436-43
Efrati, E; Tocco, G; Eritja, R et al. (1997) Abasic translesion synthesis by DNA polymerase beta violates the ""A-rule"". Novel types of nucleotide incorporation by human DNA polymerase beta at an abasic lesion in different sequence contexts. J Biol Chem 272:2559-69
David, P; Efrati, E; Tocco, G et al. (1997) DNA replication and postreplication mismatch repair in cell-free extracts from cultured human neuroblastoma and fibroblast cells. J Neurosci 17:8711-20
Sibghat-Ullah; Gallinari, P; Xu, Y Z et al. (1996) Base analog and neighboring base effects on substrate specificity of recombinant human G:T mismatch-specific thymine DNA-glycosylase. Biochemistry 35:12926-32
Law, S M; Eritja, R; Goodman, M F et al. (1996) Spectroscopic and calorimetric characterizations of DNA duplexes containing 2-aminopurine. Biochemistry 35:12329-37
Bruck, I; Woodgate, R; McEntee, K et al. (1996) Purification of a soluble UmuD'C complex from Escherichia coli. Cooperative binding of UmuD'C to single-stranded DNA. J Biol Chem 271:10767-74
Petruska, J; Arnheim, N; Goodman, M F (1996) Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases. Nucleic Acids Res 24:1992-8
Petruska, J; Goodman, M F (1995) Enthalpy-entropy compensation in DNA melting thermodynamics. J Biol Chem 270:746-50

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