Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
J Mol Biol. 2010 Apr 2;397(3):664-76. doi: 10.1016/j.jmb.2010.01.057. Epub 2010 Feb 1.
We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso) and compared in vitro priming kinetics with those of the eukaryotic-type primase (PriSL) also found in Sso. SsoDnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominate primer length was 13 bases, although less abundant products are present. SsoDnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant. Mutation of the conserved glutamate in the active site severely inhibited priming activity, suggesting a functional homology with E. coli DnaG. SsoDnaG was also found to have a greater than fourfold faster rate of DNA priming over that of SsoPriSL under optimal in vitro conditions. The presence of both enzymatically functional primase families in archaea suggests that the DNA priming role may be shared on leading or lagging strands during DNA replication.
我们对来自极端嗜热古菌嗜酸热硫化叶菌（Sulfolobus solfataricus）的类似细菌的 DnaG 引发酶进行了生化特性分析，并与同样存在于嗜酸热硫化叶菌中的真核型引发酶（PriSL）进行了体外引发动力学比较。SsoDnaG 表现出与高温引发一致的金属依赖性和温度依赖性特征。引物产物的分布是离散的，但与同源大肠杆菌 DnaG 产生的引物产物的分布高度相似。主要的引物长度为 13 个碱基，尽管存在较少的丰度产物。发现 SsoDnaG 以二聚体的形式与 DNA 结合，具有适度的解离常数，具有协同性。活性位点中保守的谷氨酸突变为引发酶活性严重抑制，表明与大肠杆菌 DnaG 具有功能同源性。在最佳体外条件下，SsoDnaG 的 DNA 引发速度比 SsoPriSL 快四倍以上。在古菌中同时存在两种具有酶活性的引发酶家族表明，在 DNA 复制过程中，DNA 引发作用可能在领头链或滞后链上共享。
