The Box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E-coli replication origin
The Box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E-coli replication origin
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DOI：
10.1074/jbc.m409695200
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发表时间：
2004-12-03
影响因子：
4.8
通讯作者：
Kaguni, JM
中科院分区：
文献类型：
--
作者：
Felczak, MM;Kaguni, JM
大肠杆菌 DnaA 蛋白从染色体起源 oriC 启动 DNA 复制，并调节该过程的频率。结构功能研究表明复制起始子包含四个结构域。基于 Aquifex aeolicus DnaA 与其他寡聚 AAA(+) 蛋白的结构相似性，提出结构域 III 在 oriC 的寡聚化中发挥作用 (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763 - 4773)。由于结构域 III 内的 Box VII 基序在 DnaA 同源物中是保守的，并且可能在寡聚化中发挥作用，因此我们通过定点诱变用丙氨酸取代了大肠杆菌 DnaA 的保守 Box VII 氨基酸，以检查该基序的作用。所有突变蛋白在体内和体外从 oriC 起始时均无活性，但它们在体内支持 RK2 质粒 DNA 复制。因此，RK2 仅需要 DnaA 功能的一个子集来进行质粒 DNA 复制。对框 VII 中精氨酸 281 (R281A) 处携带丙氨酸取代的突变体 DnaA 的生化研究表明，它在 oriC 质粒的体外复制中无活性，但这种缺陷并不是由于未能与 DnaB-DnaC 复合物中的 ATP、DnaB 或 oriC 结合。由于突变体 DnaA 在 oriC 的链打开中也具有活性，而 DnaB 无法与该解旋区域结合，因此开放结构本身不足以加载 DnaB 解旋酶。我们的结果表明，突变体无法形成稳定的寡聚 DnaA-oriC 复合物，而这是加载 DnaB 所需的。
Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA(+) proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. ( 2002) EMBO J. 21, 4763 - 4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.