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.
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