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DNA Synthesis by The Isolated Nuclear Matrix and Its Regulatory Mechanism

离体核基质合成DNA及其调控机制

基本信息

批准号：
03833012
负责人：
MUROFUSHI Kimiko
金额：
$ 1.09万
依托单位：
Ochanomizu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

The nuclear matrix plays a role as the site of DNA replication in eukaryotic cells. It was found that multi-enzyme complexes including DNA polymerase alpha, one of the replicative enzymes of eukaryotes, were associated with the matrix, and the matrix isolated from a true slime mold, Physarum polycephalum, was able to catalyze the formation of replication intermediates in vitro. Similar stimulation occurred in the nuclear matrices isolated from HeLa cells and rat ascites hepatoma cells. DNA polymerase alpha bound to a MAP-immobilized column, whereas preincubation of the enzyme with unbound MAPs prevented binding to the column. Kinetic analyzes revealed that MAPs decreased the Km value of the polymerase for deoxyribonucleoside triphosphates, irrespective of the species of template-primer. Product analyzes showed that MAPs did not alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis. These findings indicate that a putative DNA replication machinery including DNA polymerase alpha exists on the matrix.MAPs, which are the general term for the accessory proteins associated with microtubules, one of the major cytoskeletal components, stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. MAPs also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. MAPs stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Kinetic analyzes showed that MAPs decreases the Km value of the matrix for deoxyribonucleoside triphosphates. Comparison of the Km values of active- and latent-type DNA replication machineries of Physarum suggested a possible role for MAPs or MAP-like proteins in DNA replication.

核基质在真核细胞中起着DNA复制位点的作用。研究发现，包括DNA聚合酶α（真核生物复制酶之一）在内的多酶复合物与基质相关，而从真正的粘菌多头绒泡菌中分离出的基质能够在体外催化复制中间体的形成。从 HeLa 细胞和大鼠腹水肝癌细胞中分离的核基质中也发生了类似的刺激。 DNA 聚合酶 α 与 MAP 固定柱结合，而将酶与未结合的 MAP 预孵育可防止与柱结合。动力学分析表明，无论模板引物的种类如何，MAP 都会降低脱氧核糖核苷三磷酸聚合酶的 Km 值。产品分析表明，MAP 不会改变聚合酶的持续合成能力，并且 Vmax 的增加被认为是由于 DNA 合成起始频率的增加。这些发现表明，基质上存在一种假定的 DNA 复制机制，包括 DNA 聚合酶 α。MAP 是与微管相关的辅助蛋白的总称，微管是主要的细胞骨架成分之一，在激活的 DNA 作为外源模板的情况下，由从多头绒泡菌中分离出的核基质催化 DNA 合成。 MAP 还刺激从细胞核中溶解的 DNA 聚合酶 α 活性，但不刺激 DNA 聚合酶 β 活性。 MAP 通过与假定的 DNA 复制机制（包括与基质结合的 DNA 聚合酶 α）相互作用来刺激 DNA 合成。动力学分析表明，MAP 降低了脱氧核糖核苷三磷酸基质的 Km 值。绒泡菌活性型和潜伏型 DNA 复制机制的 Km 值的比较表明 MAP 或 MAP 样蛋白在 DNA 复制中可能发挥作用。