Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.
Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.
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DOI：
10.1016/s0021-9258(17)41829-1
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发表时间：
1994-02
期刊：
影响因子：
--
通讯作者：
K. Tõugu;H. Peng;K. Marians
中科院分区：
文献类型：
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作者：
K. Tõugu;H. Peng;K. Marians
在Okazaki片段合成周期中，Primase在控制滞后链上所需的一系列事件中起着关键作用。为了开始探索复制分叉处Primase功能所需的蛋白质-蛋白质相互作用，我们使用有限的胰酶分解将Primase分离为两个功能结构域，一个49 kDa的N-末端结构域(P49)和一个16 kDa的羧基-末端结构域(P16)。P49在以携带噬菌体G4复制起点的M13噬菌体DNA为模板的复制试验中保持了启动酶活性，但在一般启动或将Phi X174单链环状(ss(C))-DNA转化为复制型(RF)的过程中失去活性，并且不能支持由Phi X型原始体蛋白和DNA聚合酶III全酶重组的复制叉处的滞后链DNA合成。另一方面，p16抑制包括复制叉解旋酶Dna B的复制反应(普通启动，Phi x174 ss(C)->；RF，以及在复制叉处)，但对那些没有作用的复制反应(M13Gori ss(C)->；RF)没有影响。这些结果表明，p49定义了催化活性所需的启动酶结构域，p16定义了与DNAB功能相互作用所需的启动酶结构域，并且是蛋白质与DNAB的相互作用将启动酶吸引到复制分叉。
Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)–>RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)–>RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.