MECHANISM OF ELONGATION OF PRIMED DNA BY DNA POLYMERASE-DELTA, PROLIFERATING CELL NUCLEAR ANTIGEN, AND ACTIVATOR-1
MECHANISM OF ELONGATION OF PRIMED DNA BY DNA POLYMERASE-DELTA, PROLIFERATING CELL NUCLEAR ANTIGEN, AND ACTIVATOR-1
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DOI：
10.1073/pnas.87.15.5672
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发表时间：
1990-08-01
影响因子：
11.1
通讯作者：
HURWITZ, J
中科院分区：
文献类型：
--
作者：
LEE, SH;HURWITZ, J
在单链DNA结合蛋白（SSB）的存在下，通过DNA聚合酶δ延伸引发的DNA模板。(polδ）依赖于ATP和两种蛋白因子，激活因子1（A1）和增殖细胞核抗原（PCNA）。我们已经检查了这些蛋白质与（dA）450的相互作用。（dT）12-18的DNA形成稳定复合物的能力。在ATP、A1、PCNA和pol δ存在下，与DNA形成稳定的复合物，可以通过凝胶过滤分离。将分离的复合物与dTTP孵育导致聚（dT）的合成。虽然ATP是形成这种复合物所必需的，但它并不是随后DNA延伸所必需的。确定了复杂形成的时间要求。发现A1首先结合，然后PCNA依赖于ATP加入到A1·DNA复合物中，而聚δ则不结合。是最后添加的。这些复合物中的每一个都可以通过凝胶过滤分离，表明它们具有高度的稳定性。PCNA与A1-SSB-包被的引发DNA的结合发生在腺苷5“-[γ-腺苷]存在时。硫代三磷酸以及ATP。然而，pol. δ.只有当后者复合物在ATP存在下形成时，才观察到对PCNA·A 1·DNA复合物的抑制作用。在37 ℃温育后形成完整的复合物。在0 ℃孵育2分钟后，没有检测到复合物。C.这些结果表明，这些蛋白质的作用方式类似于辅助蛋白，在T4噬菌体DNA聚合酶和大肠杆菌DNA聚合酶III催化的延伸反应中发挥关键作用。
In the presence of a single-stranded-DNA-binding protein (SSB), the elongation of primed DNA templates by DNA polymerase .delta. (pol .delta.) is dependent on ATP and two protein factors, activator 1 (A1) and proliferating cell nuclear antigen (PCNA). We have examined the interaction of these proteins with (dA)450.cntdot.(dT)12-18 by measuring their ability to form stable complexes with this DNA. In the presence of ATP, A1, PCNA, and pol .delta. formed a stable complex with DNA that could be isolated by gel filtration. Incubation of the isolated complex with dTTP resulted in the synthesis of poly(dT). While ATP was required for the formation of this complex, it was not required for the subsequent elongation of DNA. The temporal requirements for complex formation were determined. A1 was found to bind first, followed by the ATP-dependent addition of PCNA to the A1.cntdot.DNA complex, while poly .delta. was added last. Each of these complexes could be isolated by gel filtration, indicating that they possessed a high degree of stability. The binding of PCNA to the A1-SSB-coated primed DNA occurred with adenosine 5''-[.gamma.-thio]triphosphate as well as ATP. However, the binding of pol .delta. to the PCNA.cntdot.A1.cntdot.DNA complex was observed only when the latter complex was formed in the persence of ATP. The complete complex was formed after incubation at 37.degree. C for 2 min, whereas no complex was detected after incubation at 0.degree. C. These results indicate that these proteins act in a manner analogous to the accessory proteins that play critical roles in the elongation reaction catalyzed by T4 phage DNA polymerase and Escherichia coli DNA polymerase III.