Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA and Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA.
Nucleic Acids Res. 2013 Dec;41(22):10254-67. doi: 10.1093/nar/gkt774. Epub 2013 Aug 29.
DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.
DnaA 是细菌中 DNA 复制的起始因子。一种名为 DnaAcos 的突变 DnaA 不同寻常，因为它对负调控有抗性。我们开发了一种遗传方法来分离其他规避调控的突变 DnaA，以扩展我们对控制复制起始机制的理解。与 DnaAcos 一样，一种携带组氨酸 202 被酪氨酸取代的突变体（H202Y）能够抵抗 datA、hda 和 dnaN（β 夹）施加的调控，并且 DnaAcos 和 H202Y 都能抵抗 Hda-β 夹复合物在体外的抑制。其他携带 G79D、E244K、V303M 或 E445K 取代的突变 DnaA 要么仅部分敏感，要么对 Hda-β 夹复合物在体外的抑制有抗性，但对体内 hda 的表达有反应。所有突变 DnaA 仍然能够与 Hda 直接相互作用。有趣的是，DnaAcos 和 DnaAE244K 与 Hda 的结合更紧密。这些突变体通过隔离 Hda，可能限制其可用于调节其他 DnaA 分子的可用性，这些分子仍然活跃，以诱导额外的 DNA 复制轮次。其他证据表明，一种携带 V292M 取代的突变体通过逃避未知调节因子的作用而过度起始。总之，我们的结果为调节大肠杆菌复制起始的机制提供了新的见解。
