Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA-ATP
Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA-ATP
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DOI：
10.1073/pnas.0400340101
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发表时间：
2004-03-02
影响因子：
11.1
通讯作者：
Leonard, AC
中科院分区：
文献类型：
--
作者：
McGarry, KC;Ryan, VT;Leonard, AC
真核生物、古细菌和真细菌中 DNA 复制的启动需要结构保守的 ATP 结合起始蛋白和原始 DNA 的相互作用来介导复制体的组装。然而，在启动的早期步骤中对 ATP 的具体要求仍不清楚。即使对于经过充分研究的大肠杆菌复制起点 oriC 也是如此，其中起始子 DnaA 的 ATP 形式对于初始 DNA 链分离是必要且充分的，但具有共有序列 5'TGTGNAT/AAA 的五个 DnaA 结合位点（R 框）以相同的亲和力结合活性 ATP-DnaA 和非活性 ADP-DnaA。通过使用硫酸二甲酯足迹法，我们最近鉴定了两个引发剂结合位点，12 和 13，序列为 5'TG/TGGATCAG/A。我们现在表明位点 12 和 13 优先结合 DnaA-ATP，并且是起始解旋所必需的。位点 3 的鸟嘌呤决定了 DnaA-ATP 的偏好，并且在两个 I 位点将该碱基更改为胸腺嘧啶允许 DnaA-ADP 结合并打开 oriC，尽管 DNA 链分离并不精确地位于富含 AT 的区域。这些观察结果表明，复制起点内的特定起始子结合位点可能是调节 DNA 链分离的 ATP 依赖性分子开关的重要决定因素。
Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia, coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5'TGTGNAT/AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, 12 and 13, with sequence 5'TG/TGGATCAG/A. We now show that sites 12 and 13 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation.