The biological consequences of oxidative and radiation-induced DNA damage include cell death, mutation and neoplastic transformation. A substantial body of evidence indicates that ionizing radiation- induced mutagenesis and carcinogenesis results from damage to DNA. Eukaryotic cells are frequently exposed to a variety of extrinsic and intrinsic agents that produce active oxygen species capable of damaging the cellular DNA. Central to our understanding of how oxidative and ionizing radiation-induced DNA damage is translated into biological endpoint is to identify and characterize those cellular components that act on damaged DNA to reverse its deleterious effects. The goal of this proposal is to gain an understanding of the role mediated by redoxyendonuclease, a DNA repair enzyme that recognizes a variety of base damage products, in the eukaryotic cellular response to oxidative and radiation- induced damage of DNA.
The specific aims are directed towards gaining an understanding of the physical properties, enzymology, genetics, and regulation of these enzymes in yeast and human cells. The yeast and human redoxyendonucleases (RE) will be purified to homogeneity or near homogeneity. Studies on the substrate specificities and mechanism of action of these enzymes will continue, utilizing enzymological and molecular biological approaches, such as DNA sequencing techniques for determining the base specificities of endonuclease cleavage. The identity of a minor UV photoproduct that is formed at UV wavelengths associated with the development of solar-induced skin cancer and is also a RE substrate will be determined by various analytical methods. The structural requirements for RE recognition of damaged DNA will be investigated by employing 2-D NMR structural analyses and computer modelling studies of oligonucleotides containing specific base damage products. A partial amino acid sequence of yeast RE will be obtained and used to construct synthetic oligonucleotides for use as hybridization probes and primers for cDNA synthesis. Monoclonal antibodies to yeast RE will also be generated. Molecular clones of yeast RE will be obtained though screening of yeast genomic libraries with oligonucleotide probes, antibodies, or hybridization probes form the E. coli endo III gene. The full- length yeast RE DNA will be sequenced and the amino acid sequence determined. The cloning the characterization of the yeast RE gene will permit study of the regulation of this enzyme in response to DNA damage in yeast cells. Gene disruption techniques will be utilized to investigate the physiological role of yeast RE. The gene probes and antibodies generated in the yeast RE studies may have utility for probing REs from other species, including humans. Once the yeast studies have been completed, we will employ similar techniques to characterize the human RE gene. A long-term goal of these studies will be to apply methods to characterize other eukaryotic enzymes involved in the repair of oxidative and radiation-induced DNA damage. By uniting biochemical, molecular biological, and genetic approches, it is anticipated that a more complete understanding of how redoxyendonuclease and subsequently, other DNA repair enzymes maintain the genetic stability of eukaryotic cell, including human cells, will emerge.
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