MULTIENZYME COMPLEX FOR DNA SYNTHESIS IN MAMMALIAN CELLS

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Deoxyribonucleic acid (DNA) possesses an innate ability to duplicate precisely. While this duplication does not require intervention of additional overriding sources of specific information, the enzymological mechanism by which it is accomplished is still not clear. Besides the mechanism of DNA replication, the control of this process, especially as related to other macromolecular synthesis and cell growth, is still poorly understood. Extension of the proposed studies should clarify these questions. In general, the knowledge gained through these studies can be used in developing new and effective strategies for cancer chemotherapy. Physical and functional interaction between the enzymes of DNA biosynthesis in mammalian cells was suggested from the observations that; a) Enzymes of DNA biosynthesis, only from the nuclei of S phase cells, co-sediment in a sucrose density gradient as a high molecular weight entity. By contrast, in G0 and G1 phase cells the enzymes were in the cytoplasm in a soluble form; b) Ribonucleoside diphosphates incorporated very efficiently into DNA or 'permeabilized' S phase, but not G1 phase, cells and c) Catalytic activity of thymidylate synthase, as measured in vivo, was critically dependent on the phase of the cell cycle and its allosteric interactions with ribonucleotide reductase, DNA polymerase-Alpha and topiosomerase. In the proposed research this multienzyme complex will be investigated further at three levels: a) Examination of the structural and functional role of DNA topoisomerase II in the assembly and maintenance of the multienzyme complex; b) Evaluation of the dynamics of enzyme associations in the presence of antineoplastic drugs, by measuring in vivo thymidylate synthase activity and deoxynucleotide pool analysis; c) Characterization of functional capabilities of the isolated complex in channeling distal precursors. Knowledge gained through these studies, particularly on the biological assay of the multienzyme complex, will then form critical basis for the purification of the complex. An understanding of the specific composition of the purified complex will be essential in achieving our ultimate objective of isolating these components in a soluble form and reconstituting the complex in vitro.