Uracil-DNA excision repair represents a major cellular defense mechanism against spontaneous and chemically induced DNA damage. Investigators propose to extend their investigations concerning the mechanism and enzymology of uracil-DNA repair in human and Escherichia coli cells. The basis for this proposal is derived from the previous investigations of mammalian and bacterial uracil-DNA glycosylases (Ung), PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein, and reconstituted human enzyme DNA repairs systems. The use of Ugi provides a novel tool for both in vitro and in vivo experiments aimed at elucidating the structure and biological functions of uracil-DNA glycosylase. Ugi is a unique protein which (i) specifically inhibits uracil-DNA glycosylases isolated from E. coli to human; (ii) completely inactivates Ung by forming a Ung-Ugi complex, and (iii) totally inhibits E. coli Ung in vivo producing an Ung-defective phenotype. The ugi gene has been cloned, sequenced, and expressed in E. coli and human fibroblasts. Furthermore, the tertiary structure of Ugi has been solved. This proposal provides a series of integrated experiments focused on determining the fidelity of uracil-DNA repair synthesis, the structure and function of uracil-DNA glycosylases, and the biological role of this enzyme in mammalian cells.
Five specific aims are set forth to achieve these objectives. (1) In the first phase of this project, the fidelity of uracil- DNA repair synthesis will be measured in HeLa cells, cell free extracts (nuclear and mitochondrial), and reconstituted uracil-DNA repair enzyme systems. This investigation will involve the M13mp2-based mutation assay for measuring uracil-DNA repair synthesis fidelity at site-specific uracil residues. (2) The DNA binding and active site of uracil-DNA glycosylase will be defined. A novel strategy that combines UV-crosslinking techniques and mass spectroscopic methods will be used to identify specific amino acids that contact DNA and uracil in the active site. (3) The tertiary structure of the Ung-Ugi complex will be solved through collaborative efforts. The solution structure will be determined using multidimensional nuclear magnetic resonance techniques and the crystal structure solved by x-ray crystallography. Structures of both the human Ung-delta84-Ugi and E. coli Ung-Ugi are anticipated. (4) Site-directed mutagenesis studies aimed at identifying critical amino acid residues in the Ugi protein will be pursued. Initial experiments will test two specific hypotheses. First, what is the role in Ung binding of the Glu and Asp residues which form an electrostatic knob on the surface of the Ugi protein, as determined from the tertiary structure? Second, are the aromatic amino acids (Tyr65 or Trp66) directly involved in inhibiting Ung activity? Site-directed mutagenesis studies will also be conducted on the E. coli Ung gene to identify amino acids located within the active site. These mutagenesis experiments will be complemented by and contribute toward an understanding of Ung/Ugi protein structures. (5) The ugi gene will be cloned and expressed in tissue culture cells. Ongoing efforts to express the ugi gene in Chinese hamster ovary cells will continue. The goal of this sub-project will be to establish a cell line that produces Ugi and inactivates uracil-DNA glycosylase in vivo. Studies to characterize the ugi phenotype of these cells should provide significant insight as to the physiological role of uracil-DNA glycosylase and uracil-DNA repair in mammalian cells. Development of an Ung-defective cell line would provide a model system for future studies of DNA repair. It is anticipated that the information gained from these investigations of uracil-DNA repair will prove to be basic and relevant to understanding the biochemical pathways that prevent mutagenesis and carcinogenesis.
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