Munsch N, Sheinin R
Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
J Cell Physiol. 1990 Sep;144(3):531-7. doi: 10.1002/jcp.1041440322.
ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.
源自野生型WT-4小鼠L细胞的ts A1S9突变细胞在DNA合成和细胞分裂方面对温度敏感（ts）。我们试图通过比较温度变化对DNA聚合酶α和DNA引发酶活性及合成随时间的影响，来确定ts A1S9细胞在非允许温度下DNA复制停滞的原因。温度升高47小时后，ts A1S9细胞的DNA聚合酶α活性被抑制了90%，而引发酶活性几乎检测不到。相比之下，在两种温度下培养的WT-4细胞以及在低允许温度下生长的ts A1S9细胞中，这两种酶的活性都增加到了一个稳定水平。通过用针对DNA聚合酶α的特异性单克隆抗体对[35S]标记的细胞进行免疫沉淀，研究了细胞周期进程中DNA聚合酶α引发酶的合成以及酶复合物的结构。我们发现，无论WT-4或ts A1S9细胞的培养温度如何，这种抗体都能从细胞提取物中沉淀出220、186、150、110、68 - 70、60和48 kDa的多肽。在38.5摄氏度下培养48小时的ts A1S9细胞中，与α聚合酶活性相关的220和186 kDa多肽比对照细胞中丰富得多，而与引发酶活性相关的60和48 kDa多肽则相应减少。因此，DNA聚合酶α的抑制不是由于186 kDa亚基合成减少，而是由于其温度失活。与最近的不对称二聚体模型一致，即聚合酶α复合物和聚合酶δ复合物在复制叉滞后和前导DNA链上协同合成，观察到的聚合酶α和引发酶含量的变化解释了ts A1S9细胞在非允许温度下DNA合成的抑制和细胞周期停滞。
