ATP negatively regulates the initiator protein of Vibrio cholerae chromosome II replication
ATP negatively regulates the initiator protein of Vibrio cholerae chromosome II replication
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DOI：
10.1073/pnas.0803904105
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发表时间：
2008-07-29
影响因子：
11.1
通讯作者：
Waldor, Matthew K.
中科院分区：
文献类型：
--
作者：
Duigou, Stephane;Yamaichi, Yoshiharu;Waldor, Matthew K.
霍乱弧菌是霍乱的病原体，它有两条环状染色体。在含有单个染色体的细菌中，染色体DNA复制的起始由DnaA介导，DnaA是一种AAA+ ATP酶，可解开复制起点。关于具有一条以上染色体的细菌中染色体复制的起始知之甚少。在这里，我们纯化了霍乱弧菌DnaA和RctB，它们与霍乱弧菌染色体II的复制有关，并表征了它们的体外活性。我们发现，RctB具有起源特异性解旋活性，可以解旋染色体II的起源（oriCIIvc），但不能解旋染色体I的起源（oriCIvc）;相反，DnaA促进oriCIvc的解旋，而不是oriCIIvc。DnaA和几种质粒起始蛋白的活性被ATP结合刺激。我们发现，RctB结合和水解ATP，即使RctB缺乏任何明显的ATP结合基序。然而，我们出乎意料地发现，ATP抑制了oriCIIvc结合活性的RctB，这表明ATP结合形式的RctB不能启动复制的染色体It。支持这一想法，我们确定了一个RctB突变体，不结合ATP，并发现这种ATP盲RctB突变体的表达在霍乱弧菌导致显着过度启动染色体II和显着抑制霍乱弧菌的生长。这些观察结果表明，两条霍乱弧菌染色体复制的许可规则不同。
Vibrio cholerae, the agent of cholera, has two circular chromosomes. In bacteria that contain a single chromosome, initiation of chromosome DNA replication is mediated by DnaA, a AAA+ ATPase that unwinds the origin of replication. There is little knowledge regarding initiation of chromosome replication in bacteria with more than one chromosome. Here, we purified V. cholerae DnaA and RctB, which have been implicated in the replication of V. cholerae chromosome II, and characterized their activities in vitro. We found that RctB has origin-specific unwinding activity and can melt the origin of chromosome II (oriCIIvc) but not the origin of chromosome I (oriCIvc); conversely, DnaA promoted the unwinding of oriCIvc and not oriCIIvc. The activity of DnaA and several plasmid initiator proteins is stimulated by ATP binding. We found that RctB bound and hydrolyzed ATP even though RctB lacks any apparent ATP-binding motifs. However, we unexpectedly found that ATP inhibited the oriCIIvc binding activity of RctB, suggesting that the ATP-bound form of RctB cannot initiate replication of chromosome It. Supporting this idea, we identified an RctB mutant that does not bind ATP and found that expression of this ATP-blind RctB mutant in V. cholerae leads to significant overinitiation of chromosome II and marked inhibition of V. cholerae growth. These observations suggest that the rules that license the replication of the two V. cholerae chromosomes differ.