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。拟议研究的延期应澄清这些问题 问题。一般来说，通过这些研究获得的知识可以是 用于开发新的有效的癌症化疗策略。 DNA生物合成酶之间的物理和功能相互作用 在哺乳动物细胞中的作用；a)酶 脱氧核糖核酸的生物合成，仅来自S期细胞的细胞核，在一种 蔗糖密度梯度是一种高分子量实体。相反， 在G0和G1期细胞中，酶以可溶性形式存在于细胞质中 形式；b)核糖核苷二磷酸非常有效地结合到DNA中 或‘渗透性’的S相，而不是G1相，细胞和c)催化 在体内测量的胸苷合成酶活性是至关重要的。 依赖于细胞周期的阶段及其变构相互作用 核糖核酸还原酶、DNA聚合酶-α和拓扑异构酶。 在拟议的研究中，将对这种多酶复合体进行研究 进一步在三个层面上：a)审查结构和功能 DNA拓扑异构酶II在细胞装配和维持中的作用 多酶复合体；b)酶缔合动力学的评价 在抗肿瘤药物存在的情况下，通过测量体内胸苷 合酶活性和脱氧核苷酸池分析；c)表征 隔离复合体向远端导流的功能能力 先驱物。 通过这些研究获得的知识，特别是关于生物 多酶复合体的分析，然后将形成关键的基础 对络合物进行提纯。对具体作文的理解 将是实现我们最终目标的关键 以可溶形式分离这些组分的目标和 在体外重建复合体。

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.