Institute of Biochemistry, University of Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany.
Nucleic Acids Res. 2010 Oct;38(19):6707-18. doi: 10.1093/nar/gkq447. Epub 2010 May 28.
The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40-370. While the N-terminal part of that minimal region (residues 47-247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248-370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension.
质粒 pRN1 编码一种多功能复制蛋白，具有引发酶、DNA 聚合酶和解旋酶活性。引发酶活性所必需的最小区域包含氨基酸残基 40-370。虽然该最小区域的 N 端部分（残基 47-247）折叠成引发酶/聚合酶结构域并具有活性位点，但 C 端部分（残基 248-370）的结构和功能尚不清楚。在这里，我们表明最小区域的 C 端部分折叠成一个具有六个螺旋的紧凑结构域，并由一个二硫键稳定。三个螺旋与细菌广谱宿主范围质粒 RSF1010 的引发酶的 C 端结构域很好地叠加。基于结构的定点突变显示，尽管在结晶构象中远离活性位点，但螺旋束结构域的 C 端螺旋对于引发酶活性是必需的。此外，我们还鉴定了 C 端结构域的突变体，这些突变体在模板结合、二核苷酸形成和 DNA 延伸前的构象变化方面存在缺陷。
