All organisms devote a large amount of metabolic energy to scavenge oxygen free radicals, prevent the incorporation of oxidatively damaged precursors into nucleic acids, and repair oxidatively induced DNA lesions. One of the most important of these cellular activities involves the enzymes that sanitize deoxynucleoside triphosphate pools and help maintain DNA replication fidelity. This activity is paramount for the viability of the organism and may be one of the most important factors in the prevention of the oxidative damage that leads to cancer. One such sanitizing protein is the pyrophosphohydrolase MutT, the original E. coli mutator, that scans the dGTP pool and hydrolyzes potentially mutagenic 8-oxo-7,8 dihydrodeoxyguanosine triphosphate (8-oxodGTP). This damaged deoxynucleoside triphosphate is the major product of oxidative attack on dNTP pools, and the activity of MutT prevents the incorporation of this altered precursor into DNA. Incorporation of 8-oxodGMP results in the formation of mutagenic oxoG:A basepairs at an astounding rate. In E. coli, a mutT- cell has 100-1,000 times more A:T to C:G transversions than a wild-type bacterium, presumably causes by a G:A mispair intermediate. The magnitude of this single genetic lesion could be extremely detrimental to humans and may be a major cause of oxidatively induced tumors. This study proposes to examine the structural biochemistry of two MutT homologues to answer enzymological and functional questions about the relation between oxidative damage to DNA precursors, DNA replication fidelity, and the promotion of human cancers. The two homologues to be examined show low sequence identity, no known active site motifs, and length heterogeneity. The human MutT protein has been purified, substrate specificity will continue to be assayed, the enzyme will be crystallized and the structure determined by X-ray crystallography. A current crystallographic model of the E. coli MutT dGTPase will be refined. Substrate/protein crystal complexes will demonstrate the rules governing substrate specificity. In addition, site directed mutants of the human enzyme will continue to be produced and analyzed by enzyme assay for alterations in activity, and by genetic complementation for in vivo function. The tissue distribution of mutT will be determined via Northern blotting, also the level of mutT expression in normal and cancerous cell lines will be determined. This section will address questions of MutT's role as an antioncogenic agent and the development of human cancer. Finally, biochemical experiments will be employed to determine whether or not MutT functions in concert with other proteins to prevent oxidatively damaged DNA precursors from being synthesized into newly replicating DNA. The identification of factors that interact with MutT will provide insight into the in vivo activity and regulation of the enzyme, and may also provide clues to the prevention of oxidatively induced cancers.