Zhang Wenke, Dillingham Mark S, Thomas Christopher D, Allen Stephanie, Roberts Clive J, Soultanas Panos
Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
J Mol Biol. 2007 Aug 10;371(2):336-48. doi: 10.1016/j.jmb.2007.05.050. Epub 2007 May 24.
The replication initiator protein RepD recruits the Bacillus PcrA helicase directly onto the (-) strand of the plasmid replication origin oriD. The 5'-phosphate group at the nick is essential for loading, suggesting that it is the RepD covalently linked to the 5'-phosphate group at the nick that loads the helicase onto the oriD. The products of the unwinding reaction were visualised by atomic force microscopy (AFM) and monitored in real time by fluorescence spectroscopy. RepD remains associated with PcrA and stimulates processive directional unwinding of the plasmid at approximately 60 bp s(-1). In the absence of RepD, PcrA retains the ability to bind to a pre-nicked oriD, but engages the 3' end of the nick and translocates 3'-5' along the (+) strand in a poorly processive fashion. Our data provide a unique insight into the recruitment of PcrA-like helicases to DNA-nick sites and the processive translocation of the PcrA motor as a component of the plasmid replication apparatus.
复制起始蛋白RepD直接将芽孢杆菌PcrA解旋酶招募到质粒复制起点oriD的（-）链上。切口处的5'-磷酸基团对于加载至关重要，这表明正是与切口处5'-磷酸基团共价连接的RepD将解旋酶加载到oriD上。通过原子力显微镜（AFM）观察解旋反应的产物，并通过荧光光谱实时监测。RepD仍然与PcrA结合，并以约60 bp s（-1）的速度刺激质粒进行持续性定向解旋。在没有RepD的情况下，PcrA保留了与预先切口的oriD结合的能力，但与切口的3'端结合，并以较差的持续性沿着（+）链3'-5'方向移位。我们的数据为将PcrA样解旋酶招募到DNA切口位点以及PcrA马达作为质粒复制装置的一个组成部分进行持续性移位提供了独特的见解。
