Kaboord B F, Benkovic S J
Pennsylvania State University, Department of Chemistry, University Park 16802, USA.
Curr Biol. 1995 Feb 1;5(2):149-57. doi: 10.1016/s0960-9822(95)00036-4.
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.
To address this tissue, 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.
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.
在噬菌体T4 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持续合成的全酶复合物。该复合物的形成依赖于44/62蛋白对ATP的水解。然而，尚不确定在DNA合成过程中，44/62蛋白是否作为该蛋白“滑动夹”的一部分与45蛋白保持结合，或者它是否仅在复合物组装时才需要。
为了解决这个问题，我们以化学计量方式在一个叉状引物/模板上组装了一个能够进行链置换合成的持续合成的T4 DNA聚合酶全酶复合物。通过将44/62蛋白滴定至亚化学计量浓度，我们发现它可以催化作用将45蛋白加载到引物/模板上，而45蛋白又与DNA聚合酶结合形成一个持续合成的复合物。形成了两种不同的复合物种类：大多数复合物高度稳定，半衰期为7分钟，而其余复合物的半衰期为0.4分钟。对蛋白质-DNA复合物进行沉淀，随后进行蛋白质印迹分析，证实这些复合物含有DNA聚合酶和45蛋白，但不含有44/62蛋白。
使用生理蛋白质浓度，我们已经表明T4蛋白滑动夹的组成仅由45蛋白组成。44/62蛋白的作用是作为分子媒人，因为它用于将45蛋白加载到DNA上，但不是持续合成复合物的必需成分。
