The mammalian DNA polymerase delta-proliferating cell nuclear antigen-template-primer complex: Molecular characterization by direct binding
The mammalian DNA polymerase delta-proliferating cell nuclear antigen-template-primer complex: Molecular characterization by direct binding
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DOI：
10.1021/bi9530649
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发表时间：
1996-06-25
期刊：
影响因子：
2.9
通讯作者：
Fisher, PA
中科院分区：
文献类型：
--
作者：
McConnell, M;Miller, H;Fisher, PA
建立了三种直接测定法，即聚丙烯酰胺 Pel 电泳带 I-贵族位移、琼脂糖凝胶电泳带迁移率位移和硝酸纤维素滤膜结合，以研究哺乳动物 DNA 聚合酶 δ (pol gamma)、增殖性核抗原 (PCNA) 和合成寡核苷酸模板引物之间形成的复合物。在所有情况下，复合物的形成都需要同时存在 pol δ、PCNA 和模板引物。此外，我们在一项此类测定中表明，形成的复合物包含每种分子成分。核酸酶保护实验表明，复合物的形成可保护模板免遭 DNase I 降解。测定 pol δ 的质量。 PCNA模板-引物复合物大小约为267 kDa，与1分子pol δ、2-3分子PCNA和1分子模板-引物的参与一致。单独的 PCNA 表现为三聚体（质量确定为约 87 kDa）。复合物可以通过酶学方法进行操作。解离速率的测量直接表明 PCNA 稳定了 pol δ、模板-引物复合物。
Three direct assays, polyacrylamide Pel electrophoresis-band I-nobility shift, agarose gel electrophoresis-band mobility shift, and nitrocellulose filter binding, were established to study complexes formed among mammalian DNA polymerase delta (pol gamma), proliferating fell nuclear antigen (PCNA), and synthetic oligonucleotide template-primers. In all contexts, complex formation requires simultaneous presence of pol delta, PCNA, and template-primer. Moreover, we showed in one such assay that the complex formed contains each molecular component. Nuclease protection experiments demonstrate that complex formation protects template from degradation by DNase I. The mass determined for the pol delta . PCNA template-primer complex was about 267 kDa, consistent with the participation of one molecule of pol delta, two or three molecules of PCNA and one molecule of template-primer. PCNA alone behaved as a trimer (mass determined to be about 87 kDa). Complex could be manipulated enzymologically. Measurement of off rates demonstrates directly that PCNA stabilizes the pol delta, template-primer complex.