5-10-methylenetetrahydrofolic-acid and 5-fluorouridine

Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil. We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR). The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region. An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations. One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones. A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity. Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate. In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme. This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil.

Topics: Amino Acid Sequence; Binding Sites; Cell Survival; Deoxyuracil Nucleotides; Drug Resistance; Enzyme Inhibitors; Escherichia coli; Humans; Kinetics; Molecular Sequence Data; Mutagenesis; Mutation; Protein Binding; Sequence Analysis, DNA; Tetrahydrofolates; Thymidylate Synthase; Uridine

Thymidylate synthase plays a central role in the biosynthesis of thymidylate, an essential precursor for DNA biosynthesis. In addition to its role in catalysis and cellular metabolism, studies from our laboratory have shown that thymidylate synthase functions as an RNA binding protein. Specifically, thymidylate synthase binds with high affinity to its own mRNA resulting in translational repression. An extensive series of experiments have now been performed to elucidate the molecular elements underlying the interaction between thymidylate synthase and its own mRNA. These studies have shed new light into the critical nucleotide sequences and/or secondary structure that are important for protein recognition. As well, studies to define the domains on the protein essential for RNA binding are currently underway. In addition to the characterization of the cis- and trans-acting elements underlying the interaction between thymidylate synthase and its own mRNA, we have recently shown that thymidylate synthase has the capacity to specifically bind in vitro and in vivo to other cellular RNA species. In this regard, thymidylate synthase interacts with the mRNAs of the c-myc onocogene and the p53 tumor suppressor gene. These two genes have been shown to play critical roles in cell cycle control, DNA biosynthesis, and apoptosis. In vitro studies reveal that the interaction of TS with these cell-cycle related mRNAs results in their translational repression. While the biological significance of these cellular RNA/TS protein interactions remains to be defined, these studies suggest a potential role for TS as a mediator in the coordinate regulation of several critical aspects of cellular metabolism.

Topics: Antineoplastic Agents; Blotting, Southern; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Fluorodeoxyuridylate; Gene Expression Regulation, Neoplastic; Humans; Polymerase Chain Reaction; Precipitin Tests; Protein Biosynthesis; RNA-Binding Proteins; RNA, Messenger; Tetrahydrofolates; Thymidylate Synthase; Tumor Cells, Cultured; Uridine; Uridine Monophosphate