DNA Synthesis by The Isolated Nuclear Matrix and Its Regulatory Mechanism

核基质在真核细胞中起着DNA复制位点的作用。研究发现，包括真核生物复制酶之一DNA聚合酶α在内的多酶复合物与基质存在关联，并且从一种真正的黏菌绒泡菌（Physarum polycephalum）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分离的核基质中也出现了类似的刺激。DNA聚合酶与map固定柱结合，而酶与未结合的map预孵育阻止了与柱的结合。动力学分析表明，无论模板引物的种类如何，MAPs都降低了脱氧核糖核苷三磷酸聚合酶的Km值。产物分析表明，MAPs并没有改变聚合酶的活性，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，一个假定的DNA复制机制，包括DNA聚合酶存在于基质上。map是与微管相关的辅助蛋白的总称，微管是细胞骨架的主要成分之一，在激活DNA作为外源模板存在的情况下，从多头绒泡菌中分离的核基质刺激DNA合成。MAPs也刺激了细胞核溶解的DNA聚合酶α的活性，但没有刺激DNA聚合酶β的活性。MAPs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，MAPs降低了三磷酸脱氧核糖核苷的基体Km值。绒泡菌活性型和潜伏型DNA复制机制Km值的比较提示MAPs或map样蛋白可能在DNA复制中起作用。

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.