ACCESSORY PROTEINS FUNCTION AS MATCHMAKERS IN THE ASSEMBLY OF THE T4 DNA-POLYMERASE HOLOENZYME
ACCESSORY PROTEINS FUNCTION AS MATCHMAKERS IN THE ASSEMBLY OF THE T4 DNA-POLYMERASE HOLOENZYME
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DOI：
10.1016/s0960-9822(95)00036-4
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发表时间：
1995-02-01
期刊：
影响因子：
9.2
通讯作者：
BENKOVIC, SJ
中科院分区：
文献类型：
--
作者：
KABOORD, BF;BENKOVIC, SJ
背景：在T4噬菌体DNA复制过程中，44/62和45辅助蛋白与DNA聚合酶结合形成一个过程全酶复合物。这种复合物的形成依赖于44/62蛋白对ATP的水解。然而，目前尚不确定的是，在DNA合成过程中，44/62蛋白是否作为45蛋白“滑动钳”的一部分保留在45蛋白中，或者是否仅需要用于复杂组装。结果：为了解决这个问题，我们在分叉引物/模板上通过化学计量学组装了一个具有链置换合成能力的T4 DNA聚合酶全酶复合物。通过将44/62蛋白滴定到亚化学计量浓度，我们已经证明它可以催化作用，将45蛋白装载到引物/模板上，进而与DNA聚合酶结合形成一个过程复合物。形成了两个不同的复杂物种。大多数配合物高度稳定，半衰期为7分钟，而其余的半衰期为0.4分钟。蛋白质-DNA复合物的沉淀，随后进行western blot分析，证实该复合物含有DNA聚合酶和45蛋白，但不含44/62蛋白。结论：使用生理蛋白浓度，我们已经证明T4蛋白滑动钳的组成完全由45蛋白组成。44/62蛋白的作用是分子媒妁之言，因为它可以将45蛋白装载到DNA上，但并不仍然是过程复合体的重要组成部分。
Background: During bacteriophage T4 DNA replication, the 44/62 and 45 accessory proteins combine with the DNA polymerase to form a processive holoenzyme complex. Formation of this complex is dependent upon ATP hydrolysis by the 44/62 protein. It is uncertain, however, whether the 44/62 protein remains with the 45 protein as part of this protein 'sliding clamp' during DNA synthesis, or whether it is required only for complex assembly.Results: To address this issue, we have stoichiometrically assembled a processive T4 DNA polymerase holoenzyme complex, capable of strand-displacement synthesis, on a forked primer/template. By titrating the 44/62 protein to substoichiometric concentrations, we have shown that it can act catalytically to load on to the primer/template the 45 protein, which, in turn, combines with the DNA polymerase to form a processive complex. Two distinct complex species are formed. most of the complexes are highly stable, with a half life of 7 minutes, whereas the remainder have a half-life of 0.4 minutes. Precipitation of the protein-DNA complexes, followed by western blot analysis, verified that the complexes contain the DNA polymerase and 45 proteins, but not the 44/62 protein.Conclusion: Using physiological protein concentrations, we have shown that the composition of the T4 protein sliding clamp consists solely of the 45 protein. The role of the 44/62 protein is that of a molecular matchmaker, in that it serves to load the 45 protein onto the DNA but does not remain an essential component of the processive complex.