Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA
Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA
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DOI：
10.1101/gad.1775809
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发表时间：
2009-05-15
影响因子：
10.5
通讯作者：
Katayama, Tsutomu
中科院分区：
文献类型：
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作者：
Fujimitsu, Kazuyuki;Senriuchi, Takayuki;Katayama, Tsutomu
在大肠杆菌中，与ADP-DNAA不同的是，ATP-DNAA可以在ORIC启动染色体复制。细胞内的ATP-DNAA水平起伏不定，在复制开始时达到峰值。然而，目前尚不清楚ATP-DNAA水平是如何随着复制周期而协调增加的。在本研究中，我们发现了两个染色体间隔区，分别称为DNAA再激活序列1(DARS1)和DNAA再激活序列2(DARS2)，它们通过核苷酸交换直接促进由ADP-DNAA再生的ATP-DNAA，从而在体外和体内促进复制起始。启动与细胞周期的协调需要DARS的活性及其调节。DASS的过量供应导致ATP-DNAA水平升高和复制启动增强，从而以一种ORIC依赖的方式抑制细胞生长。DASS的缺失会导致ATP-DNAA水平的下降和复制启动的抑制，这可以导致温度敏感突变体DNAA的合成致死性，以及由于缺乏SEQA或数据而抑制过启动，负启动调节剂。DNAS带有一簇DNAA结合位点。DNAA分子在DARS1上形成特定的同源多聚体，这会导致原核之间的特定相互作用，降低它们与ADP的亲和力。我们的发现揭示了一种新的调控途径，它通过Dna A重新激活来促进染色体复制的启动。
In Escherichia coli, ATP-DnaA, unlike ADP-DnaA, can initiate chromosomal replication at oriC. The level of cellular ATP-DnaA fluctuates, peaking at around the time of replication initiation. However, it remains unknown how the ATP-DnaA level increases coordinately with the replication cycle. In this study, we show that two chromosomal intergenic regions, herein termed DnaA-reactivating sequence 1 (DARS1) and DnaA-reactivating sequence 2 (DARS2), directly promote regeneration of ATP-DnaA from ADP-DnaA by nucleotide exchange, resulting in the promotion of replication initiation in vitro and in vivo. Coordination of initiation with the cell cycle requires DARS activity and its regulation. Oversupply of DARSs results in increase in the ATP-DnaA level and enhancement of replication initiation, which can inhibit cell growth in an oriC-dependent manner. Deletion of DARSs results in decrease in the ATP-DnaA level and inhibition of replication initiation, which can cause synthetic lethality with a temperature-sensitive mutant dnaA and suppression of overinitiation by the lack of seqA or datA, negative regulators for initiation. DARSs bear a cluster of DnaA-binding sites. DnaA molecules form specific homomultimers on DARS1, which causes specific interactions among the protomers, reducing their affinity for ADP. Our findings reveal a novel regulatory pathway that promotes the initiation of chromosomal replication via DnaA reactivation.