The overall goal of this proposal is to continue work on the characterization of enzymes involved in nucleotide metabolism. Also included are initial studies that attempt to elucidate the mechanisms responsible for regulated fluctuation of certain enzyme activities with the DNA synthesis phase of the cell cycle. These studies may eventually aid in determining the efficacy of anticancer agents such as methotrexate, cytosine arabinoside and related analogs. Futhermore, uncovering basic information of how the cell regulates the synthesis of the machinery necessary for its own proliferation may lead to an understanding of growthcontrolling elements. The aberrant growth regulation of a cancer cell may then begin to be understood. This research proposal has three main aspects: 1. Further characterization of dUTPase, the enzyme responsible for dUTP hydrolysis in the cell. This will entail the use of monoclonal antibodies already generated against the dUTPase enzyme derived from HeLa S3 cells. An attempt will be made to elucidate the reason for post-translational phosphorylation of the dUTPase protein. 2. Generations of monoclonal antibodies against SDS polyacrylamide gel purified enzyme that apparently hydrolyzes dCTP within the cell. These antibodies will then be used to characterize this dCTPase enzyme in terms of its structure and function. 3. Analysis of thymidine kinase and dUTPase from human cells in terms of whether posttranslational modification occurs as a cell cycle dependent modification, coordinate with their enzyme activities. This aspect of the proposal will utilize methods of cell synchronization, immunoprecipitation and enzymatic assays for the analysis of the proteins under study.

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National Cancer Institute (NCI)
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Experimental Therapeutics Subcommittee 2 (ET)
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University of Medicine & Dentistry of NJ
Schools of Osteopathy
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Muller-Weeks, S; Mastran, B; Caradonna, S (1998) The nuclear isoform of the highly conserved human uracil-DNA glycosylase is an Mr 36,000 phosphoprotein. J Biol Chem 273:21909-17
Ladner, R D; Caradonna, S J (1997) The human dUTPase gene encodes both nuclear and mitochondrial isoforms. Differential expression of the isoforms and characterization of a cDNA encoding the mitochondrial species. J Biol Chem 272:19072-80
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Ladner, R D; McNulty, D E; Carr, S A et al. (1996) Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase. J Biol Chem 271:7745-51
Muller-Weeks, S J; Caradonna, S (1996) Specific association of cyclin-like uracil-DNA glycosylase with the proliferating cell nuclear antigen. Exp Cell Res 226:346-55
Muller, S J; Caradonna, S (1993) Cell cycle regulation of a human cyclin-like gene encoding uracil-DNA glycosylase. J Biol Chem 268:1310-9
Worrad, D M; Caradonna, S (1993) The herpes simplex virus type 2 UL3 open reading frame encodes a nuclear localizing phosphoprotein. Virology 195:364-76
Muller, S J; Caradonna, S (1991) Isolation and characterization of a human cDNA encoding uracil-DNA glycosylase. Biochim Biophys Acta 1088:197-207
Lirette, R; Caradonna, S (1990) Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection. J Cell Biochem 43:339-53

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