Edgar Rotem, Biek Donald, Yarmolinsky Michael
Laboratory of Biochemistry, National Cancer Institute, NIH 37 Convent Drive, Bethesda, MD 20892-4255, USA.
Mol Microbiol. 2006 Jan;59(1):276-87. doi: 10.1111/j.1365-2958.2005.04933.x.
ParA and ParB proteins and cis-acting site, parS, are required to partition plasmid P1 faithfully to daughter cells. The process may initiate from plasmids paired by ParB at which recruited ParA then acts to effect the separation. We previously reported evidence for ParB-mediated pairing of parS sites on plasmids in the absence of ParA. In DNA gyrase-inhibited cells, the pairing prevented diffusion of transcription-generated positive supercoils. This supercoil trapping was almost entirely in plasmid dimers, where the location of the parS sites in cis facilitated their pairing. Here we show that the addition of ParA blocked supercoil diffusion also in plasmid monomers. The possibility that this result is attributed to an enhancement by ParA of ParB-mediated pairing in trans is consistent with our finding that ParA appeared to partially suppress the pairing defect of two mutant ParB proteins. However, enhanced pairing alone could not account for the diffusion barrier in plasmid monomers; it was manifest in monomers even when they were largely devoid of partners in the same cell. Apparently, ParA altered the ParB-parS complex such that it could no longer swivel, most likely by anchoring it, a reaction of probable relevance to partition.
ParA和ParB蛋白以及顺式作用位点parS是质粒P1准确分配到子代细胞所必需的。该过程可能起始于由ParB配对的质粒，随后募集的ParA发挥作用实现分离。我们之前报道了在没有ParA的情况下，ParB介导质粒上parS位点配对的证据。在DNA促旋酶抑制的细胞中，这种配对阻止了转录产生的正超螺旋的扩散。这种超螺旋捕获几乎完全发生在质粒二聚体中，其中顺式排列的parS位点的位置促进了它们的配对。在这里我们表明，添加ParA也能在质粒单体中阻止超螺旋扩散。这一结果归因于ParA增强了反式的ParB介导的配对，这一可能性与我们的发现一致，即ParA似乎部分抑制了两种突变ParB蛋白的配对缺陷。然而，单纯增强配对并不能解释质粒单体中的扩散屏障；即使在同一细胞中质粒单体基本没有配对伙伴时，扩散屏障在单体中依然存在。显然，ParA改变了ParB-parS复合物，使其不再能够旋转，很可能是通过将其锚定，这一反应可能与分配有关。
