MULTIENZYME COMPLEX FOR DNA SYNTHESIS IN MAMMALIAN CELLS

脱氧核糖核酸(DNA)具有先天的复制能力 正是如此。虽然这种复制不需要干预 其他压倒一切的特定信息来源，酶学 实现这一目标的机制尚不清楚。除了 DNA复制的机制，对这一过程的控制，特别是作为 与其他大分子合成和细胞生长相关的，仍然很差 明白了。 DNA生物合成酶之间的物理和功能相互作用 从观察到的结果可以看出：(1)酶 蔗糖中DNA复制和前体生物合成共沉淀物的研究 密度梯度作为一个高分子量实体。这个复杂的分数 仅从S期细胞的细胞核中获得。它不在G0中 或G1期细胞，酶位于细胞质中； 核糖核苷二磷酸非常有效地整合到DNA中 渗透的S相，而不是G1相，以及(3)催化活性 体内测定的胸苷酸合酶严重依赖于 细胞周期的时相及其与核糖核苷酸的变构相互作用 还原酶、DNA聚合酶α和拓扑异构酶。 在这项研究中，这种多酶复合体将在 三个层次：(1)研究DNA的结构和功能作用 拓扑异构酶II在多酶的组装和维持中的作用 复合体；(2)评估酶联合的动力学。 通过体内胸苷酸合酶检测抗肿瘤药物的存在 活性和脱氧核酸池分析；(3)表征 隔离复合体向远端导流的功能能力 先驱物。(一)

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)