Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences
Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences
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DOI：
10.1074/jbc.m513654200
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发表时间：
2006-07-28
影响因子：
4.8
通讯作者：
Komano, Teruya
中科院分区：
文献类型：
--
作者：
Gyohda, Atsuko;Zhu, Shujuan;Komano, Teruya
质粒R64的shufflon由四个DNA片段组成，它们被七个sfx重组位点分开并位于两侧。任何反向sfx序列之间的Rci介导的重组导致DNA片段独立地或成组地反向。R64 shufflon选择编码IV型菌毛粘附素的七个pilV基因中的一个，其中N-末端区域是恒定的，而C-末端区域是可变的。R64 sfx序列是不对称的。sfx中心区和右臂序列是保守的，但左臂序列不是。在这里，我们构建了一个对称的sfx序列，其中sfx左臂序列被改变为右臂序列的反向重复序列，并使人工shufflon片段携带对称sfx序列的反向或正向。对称的sfx序列表现出最高的倒位频率在shufflon段两侧的两个倒位sfx序列。观察到侧翼为两个直接对称sfx序列的shufflon片段的Rci依赖性缺失，表明R64 sfx序列的不对称性抑制直接sfx序列之间的重组。此外，还观察到对称sfx序列之间的分子间重组。Rci的额外的C-末端结构域被证明对于使用不对称sfx序列的R64 shufflon的倒位是必需的，但对于使用对称sfx序列的重组不是必需的，这表明Rci的C-末端区段有助于Rci与不对称sfx序列的结合。缺少C-末端结构域的Rci蛋白与对称sfx序列的两个臂结合，但仅与不对称sfx序列的右臂结合。
The shufflon of plasmid R64 consists of four DNA segments separated and flanked by seven sfx recombination sites. Rci-mediated recombination between any inverted sfx sequences causes inversion of the DNA segments independently or in groups. The R64 shufflon selects one of seven pilV genes encoding type IV pilus adhesins, in which the N-terminal region is constant, while the C-terminal regions are variable. The R64 sfx sequences are asymmetric. The sfx central region and right arm sequences are conserved, but left arm sequences are not. Here we constructed a symmetric sfx sequence, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence and made artificial shufflon segments carrying symmetric sfx sequences in inverted or direct orientations. The symmetric sfx sequence exhibited the highest inversion frequency in a shufflon segment flanked by two inverted sfx sequences. Rci-dependent deletion of a shufflon segment flanked by two direct symmetric sfx sequences was observed, suggesting that asymmetry of R64 sfx sequences inhibits recombination between direct sfx sequences. In addition, intermolecular recombination between symmetric sfx sequences was also observed. The extra C-terminal domain of Rci was shown to be essential for inversion of the R64 shufflon using asymmetric sfx sequences but not essential for recombination using symmetric sfx sequences, suggesting that the Rci C-terminal segment helps the binding of Rci to asymmetric sfx sequences. Rci protein lacking the C-terminal domain bound to both arms of symmetric sfx sequence but only to the right arm of asymmetric sfx sequence.