Shrimankar P, Stordal L, Maurer R
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960.
J Bacteriol. 1992 Dec;174(23):7689-96. doi: 10.1128/jb.174.23.7689-7696.1992.
The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination.
大肠杆菌的dnaB基因编码一种必需的DNA复制酶。该酶利用ATP水解为ADP + P(i)所产生的能量，在DNA聚合酶之前解开双链DNA。在此过程中，它间歇性地刺激引发酶合成冈崎片段的RNA引物。为了更好地理解DnaB功能的这些及其他方面的结构基础，我们启动了对突变型DnaB蛋白的研究。在此，我们报告了一种突变型DnaB蛋白（RC231）的纯化和特性，该蛋白在第231位残基处含有半胱氨酸而非精氨酸。与野生型DnaB一样，突变蛋白能形成稳定、正确折叠的结构，允许六个亚基缔合形成六聚体。此外，突变蛋白能与ATP、不可水解的ATP类似物腺苷 - 5'-O-(3 - 硫代三磷酸)（ATPγS）、ADP和聚(dT)相互作用，并刺激引发酶的作用。然而，它在染色体复制起点处的ATP水解、解旋酶活性和复制活性方面存在严重缺陷。另外，虽然野生型DnaB与ATP的一般引发反应会引发短引物的合成，但与DnaB和ATPγS或与RC231和ATP或ATPγS的反应则刺激了长得多的引物的合成。基于这些观察结果，我们认为在一般引发过程中，引发酶在整个引物合成过程中直接与DnaB相互作用，直到DnaB从DNA上解离或DnaB进行ATP水解破坏这种相互作用并导致引物终止。
