Borrell L, Yang J, Pittard A J, Praszkier J
Department of Microbiology and Immunology, The University of Melbourne, Vic. 3010, Australia.
Plasmid. 2006 Sep;56(2):88-101. doi: 10.1016/j.plasmid.2006.04.002. Epub 2006 Jun 13.
The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.
对IncL/M质粒pMU604的复制起点进行了分析，以确定对起始蛋白结合和起点活性重要的序列。一个三次重复的序列基序5'-NANCYGCAA-3'被确定为起始蛋白RepA的结合位点（RepA框）。RepA框的所有三个拷贝对于体内活性都是必需的，并且RepA与这些框的结合似乎具有协同作用。位于RepA框紧邻上游的一个DnaA R框（框1），在pMU604的RepA启动复制起始过程中，对于DnaA的募集不是必需的，除非位于起点远端的DnaA R框（框3）已失活。然而，当起始蛋白RepA来自远缘相关质粒pMU720时，DnaA R框1对于将DnaA募集到pMU604的复制起点很重要。一个打乱DnaA R框1和3的突变以及一个打乱DnaA R框1、3和4的突变，对pMU720的RepA起始复制的影响比对pMU604的RepA起始复制的影响更具有害性。在没有DnaA的情况下，两种Rep蛋白都不能从pMU604的起点启动复制，这表明它们之间的差异可能在于将DnaA募集到这个起点的机制。DnaA蛋白增强了pMU604的RepA的结合和起点解旋活性，但在没有其他蛋白质的情况下，似乎无法与携带pMU604复制起点的线性DNA片段结合。
