The research involves studies of the mechanisms of the reactions catalyzed by the adenylate cyclase from Salmonella typhimurium and three enzymes involved in dark DNA repair processes in Escherichia coli. The bacterial adenylate cyclase is intimately involved in the process of catabolite repression, and we have demonstrated that this enzyme contains a discrete catalytic domain. We will use recombinant DNA techniques to facilitate purification of the intact enzyme as well as the catalytic domain; with the purified enzymes we will undertake kinetic and enzymological studies of the reaction mechanism. Dark DNA repair involves the correction of chemically produced lesions in DNA; we will focus our attention on the mechanisms of three enzymes that are involved in these repair processes: 1) Uracil-DNA glycosylase catalyzes the hydrolysis of the glycosidic bonds in deoxyuridine residues that arise by the spontaneous hydrolysis of the 4-amino group of cytosine. Our studies will employ sequence defined synthetic oligonucleotides as substrates, and we will use kinetic isotope effect studies to ascertain whether catalysis involves the formation of a covalent adduct with the uridine residue; we will also use primer directed mutagenesis to ascertain the importance of an essential cysteine residue in catalysis. 2) Bacteriophage T4 UV endonuclease V initiates the repair of apurinic/apyrimidinic (AP) sites in DNA (such as those produced by uracil-DNA glycosylase) by cleavage of the phosphodiester backbone on the 3'-side of the AP site. Our studies will employ sequence defined synthetic oli8gonucleotides as substrates, and we will use NMR methods to identify the reaction products. Our work on this enzyme will also involve recombinant DNA techniques to facilitate purification of large amounts of the enzyme. 3) 06 Methylguanine-DNA methyltransferase removes mutagenic alkyl groups from the 06 position of guanine, thereby preventing base pair transversions. This reaction involves the irreversible transfer of the methyl group to a cysteine residue in the methyltransferase, and we will determine the stereochemical course of this reaction using sequence defined synthetic oligonucleotides as substrates; the required configurational analyses will be accomplished by 1H NMR spectroscopy.
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