Masai H, Arai K I
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Eur J Biochem. 1995 Jun 1;230(2):384-95. doi: 10.1111/j.1432-1033.1995.tb20573.x.
The ABC primosome is assembled from DnaA, DnaB and DnaC proteins at a stem-and-loop structure containing a dnaA box within its stem (A site), and catalyses primer RNA synthesis for DNA chain elongation. The DnaA protein can bind to the A site and the A-site-DnaA-protein complex can be isolated by gel-filtration chromatography in the absence of nucleotides. Mutations within the dnaA box completely abolish the binding of DnaA protein. Point mutations within the stem region outside the dnaA box also severely reduce the affinity of DnaA protein for the A site. These results indicate that not only the dnaA box but also other nucleotides and/or secondary structure features of the stem are important for proper recognition of the A site by DnaA protein. The preprimosome, which is able to synthesize RNA primers upon addition of primase, can be isolated by gel-filtration chromatography in the presence of ATP or adenosine 5'-[gamma-thio]triphosphate, a non-hydrolyzable analogue of ATP. The preprimosome can translocate along Escherichia coli single-stranded-DNA-binding protein-coated single-stranded DNA, utilizing the energy released by hydrolysis of ATP, as indicated by its helicase activity. dATP, as well as dCTP, can support the helicase activity of the preprimosome to some extent, while they are inert in helicase assays with DnaB protein in the absence of E. coli single-stranded DNA-binding protein. In keeping with this result, the isolated preprimosome, which appears to contain DnaA and DnaB proteins, is capable of hydrolyzing dATP as well as ATP and GTP. In a reconstituted replication assay, addition of excess dATP restores replication activities which have been inhibited by addition of adenosine 5'-[gamma-thio]triphosphate. The ability of dATP to support helicase and replicative activities of the ABC primosome indicates that the formation of the complex somehow modulates the structures of its component(s) so that they can utilize otherwise inert nucleotides. On the basis of these results, a scheme for the assembly of the ABC primosome at the A site is presented.
ABC引发体由DnaA、DnaB和DnaC蛋白在其茎部含有dnaA框的茎环结构（A位点）处组装而成，并催化引物RNA合成以进行DNA链延伸。DnaA蛋白可结合到A位点，且在无核苷酸的情况下，A位点-DnaA-蛋白复合物可通过凝胶过滤色谱法分离。dnaA框内的突变会完全消除DnaA蛋白的结合。dnaA框外茎区域内的点突变也会严重降低DnaA蛋白对A位点的亲和力。这些结果表明，不仅dnaA框，而且茎的其他核苷酸和/或二级结构特征对于DnaA蛋白正确识别A位点也很重要。在加入引发酶后能够合成RNA引物的前引发体，可在存在ATP或腺苷5'-[γ-硫代]三磷酸（ATP的一种不可水解类似物）的情况下通过凝胶过滤色谱法分离。前引发体可利用ATP水解释放的能量沿着大肠杆菌单链DNA结合蛋白包被的单链DNA移位，这由其解旋酶活性表明。dATP以及dCTP在一定程度上可支持前引发体的解旋酶活性，而在无大肠杆菌单链DNA结合蛋白的情况下，它们在DnaB蛋白的解旋酶测定中是惰性的。与此结果一致，分离出的似乎含有DnaA和DnaB蛋白的前引发体能够水解dATP以及ATP和GTP。在重组复制测定中，加入过量dATP可恢复因加入腺苷5'-[γ-硫代]三磷酸而被抑制的复制活性。dATP支持ABC引发体的解旋酶和复制活性的能力表明，复合物的形成以某种方式调节其组分的结构，使其能够利用原本惰性的核苷酸。基于这些结果，提出了ABC引发体在A位点组装的方案。
