The long-term objective of this project is to better understand the relationship between the processing of DNA damage and the location of that damage in various regions of the genome. Although the general pathways for excision repair of various classes of structural defects in mammalian cells have been worked out, the """"""""average"""""""" response of the entire genome has been assayed in most repair studies. Therefore, it is important to understand in detail how the excision repair responses might reflect unique features or regions of the genome, such as chromosomal location or status of expression of structural genes. The overall experimental strategy is to simplify the analysis by using defined DNA sequences so that processing of damage can be analyzed at the molecular level. I will use as probe sequences the unexpressed alpha DNA sequence, the expressed and integrated prokaryotic gpt and neo genes and the expressed metallothionein genes. The following experimental approaches are planned: (1) Having found that DNA damage is preferentially repaired in the integrated and actively transcribed pSV2-gpt compared with the bulk of the genome while repair is deficient in the alpha DNA sequences in the same monkey cells, I will further develop the use of defined sequences as probes for DNA damage and repair by testing whether the preferential repair of pSV2-gpt is a property of integrated the plasmid or whether the endogenous metallothionein genes are also preferentially repaired. (2) I have demonstrated the physical resolution of DNA fragments containing bromodeoxyuridine (BrdUrd) labeled repair patches using a monoclonal antibody which binds to BrdUrd. However, the antibody currently available was produced against the nucleoside 5-iododeoxyuridine. To maximize the sensitivity of the separation of repaired from unrepaired DNA and to obtain antibodies with greater affinity for BrdUrd in DNA, additional monoclonal antibodies will be produced specifically against BrdUrd in DNA. (3) Since most (85-90%) human cancers originate from epithelial cells, the processing of DNA damage in the metallothionein genes in human mammary epithelial cell cultures will be studied. Excision repair of various types of DNA damage, e.g., thymine glycols produced by ionizing radiation, pyrimidine dimers formed by ultraviolet radiation and bulky chemical adducts produced by benzo(a)pyrene or aflatoxin B1, will be measured in normal human epithelial cell cultures and cell lines derived from benzo(a)pyrene treated primary cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA040453-01
Application #
3180421
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1985-07-01
Project End
1988-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Shackelford, R E; Innes, C L; Sieber, S O et al. (2001) The Ataxia telangiectasia gene product is required for oxidative stress-induced G1 and G2 checkpoint function in human fibroblasts. J Biol Chem 276:21951-9
Leadon, S A (2000) Transcription-coupled repair: a multifunctional signaling pathway. Cold Spring Harb Symp Quant Biol 65:561-6
Xing, J Z; Lee, J; Leadon, S A et al. (2000) Measuring DNA damage using capillary electrophoresis with laser-induced fluorescence detection. Methods 22:157-63
Avrutskaya, A V; Leadon, S A (2000) Measurement of oxidative DNA damage and repair in specific DNA sequences. Methods 22:127-34
Le Page, F; Kwoh, E E; Avrutskaya, A et al. (2000) Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome. Cell 101:159-71
Meyer, K M; Hess, S M; Tlsty, T D et al. (1999) Human mammary epithelial cells exhibit a differential p53-mediated response following exposure to ionizing radiation or UV light. Oncogene 18:5795-805
Cressman, V L; Backlund, D C; Avrutskaya, A V et al. (1999) Growth retardation, DNA repair defects, and lack of spermatogenesis in BRCA1-deficient mice. Mol Cell Biol 19:7061-75
Leadon, S A; Avrutskaya, A V (1998) Requirement for DNA mismatch repair proteins in the transcription-coupled repair of thymine glycols in Saccharomyces cerevisiae. Mutat Res 407:177-87
Gowen, L C; Avrutskaya, A V; Latour, A M et al. (1998) BRCA1 required for transcription-coupled repair of oxidative DNA damage. Science 281:1009-12
Le, X C; Xing, J Z; Lee, J et al. (1998) Inducible repair of thymine glycol detected by an ultrasensitive assay for DNA damage. Science 280:1066-9

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