FURTHER CHARACTERIZATION OF HELA DNA-POLYMERASE-EPSILON
FURTHER CHARACTERIZATION OF HELA DNA-POLYMERASE-EPSILON
复制标题
DOI：
10.1074/jbc.270.14.7799
复制
发表时间：
1995-04-07
影响因子：
4.8
通讯作者：
LINN, S
中科院分区：
文献类型：
--
作者：
CHUI, G;LINN, S
利用Mono-S快速蛋白液相色谱分离纯化HeLa细胞中的DNA聚合酶epsilon(Polepsilon)，使其与Polα完全分离。纯化的Polepsilon制剂在变性的聚丙烯酰胺凝胶上有两个>；200和55 kDa的多肽和少量的122 kDa的活性蛋白水解物。POL epsilon(以及POLα和Delta)在100-150 mM谷氨酸钾和15 mM氯化镁中活性最佳。复制因子RF-A和RF-C、增殖细胞核抗原和大肠杆菌单链DNA结合蛋白对该制剂的Polepsilon活性、加工能力和底物专一性没有显著影响。研究了在正常人成纤维细胞和HeLa细胞中催化亚基(>；200 kDa)的polepsilon转录本的大小。检测到7.7kb的转录本，在增殖的HeLa细胞中的比例是在静止的HeLa细胞中的5-16倍。紫外线照射后HeLa细胞和成纤维细胞Polepsilon转录水平无明显差异。小鼠多克隆抗血清与葡萄球菌蛋白A融合形成144个氨基酸的Pol epsilon多克隆抗血清，免疫印迹法特异性识别Pol epsilon的催化亚基，但不识别Polα、β或Delta的催化亚基。此外，用柱纯化的小鼠多克隆抗血清能够识别和中和polepsilon，并制备了一种能够特异性识别polepsilon催化亚基的鼠单抗。
DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of >200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (>200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.