We propose to extend our investigations on the enzymology of uracil-DNA excision repair and develop a model system for studying its physiological significance in human cells. This proposal is derived from our previous investigations on mammalian uracil-DNA glycosylases (Ung) and on the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein. Three attributes make Ugi an exceptional tool for probing the structure and physiological importance of Ung in uracil-DNA repair. (1) Ugi specifically inhibits all biological sources of Ung tested (E. coli to human). (2) Ugi completely inactivates E. coli Ung both in vitro and in vivo; the latter causes an Ung-defective phenotype. (3) the ugi gene has been cloned, sequenced and overexpressed in E. coli. In the first phase of this project the major objective will be to elucidate the molecular interactions of Ugi- Ung binding. Using cloned and overproduced Ugi and Ung, highly purified preparations will be used to define the affinity, stoichiometry and reversibility of the Ugi-Ung complex. Specific amino acids involved in complex formation will be identified using a combination of protein modification and oligonucleotide-directed mutagenesis techniques. An affinity column will be constructed by attaching Ugi to a support matrix for rapid purification and characterization of various uracil-DNA glycosylases (wild-type and mutants). As a second approach to defining the structure and function of Ugi, a study will be initiated to compare the bacteriophage PBS2 and T5 uracil-DNA glycosylase inhibitor proteins and genes. This will involve cloning and sequencing the T5 ugi gene and comparative biochemical characterization of each inhibitor. Using random oligonucleotide-directed mutagenesis, ugi mutants will be isolated and Ugi purified to apparent homogeneity. Critical amino acid residues required for inhibitor function will be identified by DNA sequencing and the properties of Ugi determined both in vitro and in vivo. Using a saturation mutagenesis technique, we will also attempt to isolate ung mutants which are defective in Ugi interaction but exhibit normal catalytic activity. Identification and characterization of these ung mutations will undoubtably aid in understanding both Ugi and Ung functions. Using high-affinity nucleic acid ligands identified by the SELEX procedure, the binding interactions with Ung in the presence or absence of Ugi will be analyzed. Oligonucleotides that preferentially bind Ung will be synthesized containing uracil residues to determine whether they are also preferred DNA substrates. Finally, the ugi gene will be cloned and expressed in tissue culture cells. The goal of this phase of the project will be to develop a human or Chinese hamster ovary cell line with defective uracil-DNA glycosylase activity. Furthermore, studies will be initiated to characterize the phenotype of these cells with respect to mutation frequency and other DNA repair related aberrations. The development of Ung-defective cell line with a defined defect in uracil-DNA repair would offer a significant advance for future studies of mammalian DNA repair. It is anticipated that the observations gained from these studies will be basic and relevant to understanding biochemical pathways for preventing mutagenesis and carcinogenesis.
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