ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA
ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA
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DOI：
10.1073/pnas.0913060107
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发表时间：
2010-03-09
影响因子：
11.1
通讯作者：
Egelman, Edward H.
中科院分区：
文献类型：
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作者：
Hui, Monica P.;Galkin, Vitold E.;Egelman, Edward H.
大多数细菌染色体都含有质粒区(PAR)的同源基因。这些基因座编码被称为PARA的ATPase，被认为有助于染色体和质粒分离所需的机械力。在霍乱弧菌中，第二染色体(ChrII)PAR基因座是chrII分离所必需的。在这里，我们发现纯化的ParA2具有与其他对位同系物相当的ATPase活性，但与许多其他对位同系物不同的是，在单独存在ATP的情况下，不能形成高分子量的复合体。相反，高分子量ParA2聚合物的形成需要DNA。电子显微镜和三维重建显示ParA2以不依赖于序列的方式在双链DNA上形成双极螺旋丝。当ParA2在ATP存在下与在没有核苷酸辅助因子的情况下聚合时，这些细丝的螺距发生了明显的变化。将ParA2的晶体结构适合于我们的细丝重建，展示了ParA2的二聚体如何与DNA结合。用三磷酸腺苷形成的细丝是左撇子，但令人惊讶的是，这些细丝在它们结合的右旋B-DNA上没有拓扑变化。结合的化学计量比是每八个碱基对对应一个二聚体，这决定了ParA2细丝的几何形状，每120埃间距左旋有4.4个二聚体。我们的发现将对理解副蛋白如何在质粒和染色体分离中发挥作用至关重要。
Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 angstrom pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.