MULTIENZYME COMPLEX FOR DNA SYNTHESIS IN MAMMALIAN CELLS

脱氧核糖核酸（DNA）具有复制的先天能力 没错 虽然这种重复不需要 其他重要的具体信息来源，酶学 实现这一目标的机制尚不清楚。 除了 DNA复制的机制，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， 明白 扩大拟议的研究应澄清这些问题， 问题. 一般来说，通过这些研究获得的知识可以 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，从以下观察中提出： DNA的生物合成，只从S期细胞的细胞核，共沉积在一个细胞中， 蔗糖密度梯度作为高分子量实体。 相比之下， 在G 0和G1期细胞中，酶以可溶性形式存在于细胞质中， 形式; B）非常有效地掺入DNA中的核糖核苷二磷酸 或“透化的”S期，但不是G1期的细胞，和 胸苷酸合成酶的活性，如在体内测量的， 依赖于细胞周期的阶段及其变构相互作用 核糖核苷酸还原酶、DNA聚合酶-α和拓扑异构酶。 在拟议的研究中，将研究这种多酶复合物 （a）审查机构的结构和职能， DNA拓扑异构酶II在组装和维持中的作用 多酶复合物; B）酶缔合动力学的评价 在存在抗肿瘤药物的情况下，通过测量体内胸苷酸 合成酶活性和脱氧核苷酸库分析; 分离复合体在引导远端中的功能能力 前体 通过这些研究获得的知识，特别是关于 多酶复合物的测定，然后将形成关键的基础， 复合物的纯化。 对具体成分的理解 将是实现我们最终目标的关键 目的是以可溶形式分离这些组分， 在体外重建复合物。

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.