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. Physical and functional interaction between the enzymes of DNA biosynthesis in mammalian cells was suggested from the observations that: (1) enzymes of DNA replication and precursor biosynthesis co-sediment in a sucrose density gradient as a high molecular weight entity. This complex fraction was obtained from the nuclei of only S phase cells. It was absent from G0 or G1 phase cells, in which the enzymes were found in the cytoplasm; (2) ribonucleoside diphosphates incorporated very efficiently into DNA of 'permeabilized' S phase, but not G1 phase, and (3) 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 topoisomerase. In this research this multienzyme complex will be investigated further at three levels: (1)\examination of the structural and functional role of DNA topoisomerase II in the assembly and maintenance of the multienzyme complex; (2) evaluation of the dynamics of enzyme associations in the presence of antineoplastic drugs, by measuring in vivo thymidylate synthase activity and deoxynucleotide pool analysis; (3) characterization of functional capabilities of the isolated complex in channeling distal precursors. (I)