Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA
Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA
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DOI：
10.1111/mmi.12525
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发表时间：
2014-03-01
影响因子：
3.6
通讯作者：
Leonard, Alan C.
中科院分区：
文献类型：
--
作者：
Kaur, Gulpreet;Vora, Mansi P.;Leonard, Alan C.
在大肠杆菌预复制复合体（pre-RC）的组装过程中，启动物dna低聚物从oriC中三个广泛分离的高亲和dna识别位点成核。寡聚体的组装由低亲和力的DNA识别位点引导，但也受到oriC中开关状构象变化的调节，这是由分别作为抑制剂和激活剂的两种DNA弯曲蛋白Fis和IHF的顺序结合介导的。尽管它们的识别位点相隔高达90bp，但Fis抑制IHF结合和弱dna相互作用，直到积累的dna取代Fis脱离oriC。目前尚不清楚高亲和力dna结合是否在远距离抑制Fis中发挥任何作用，也不清楚是否所有高亲和力dna识别位点在低聚物形成中发挥同等作用。为了研究这些问题，我们开发了起源选择性重组方法来突变大肠杆菌染色体oriC。我们发现，尽管低聚物是在没有任何单个高亲和力dna结合位点的情况下组装的，但外周位点dna结合的缺失消除了Fis的抑制，并使Fis和IHF的结合变得必不可少。我们提出了一个模型，其中DNA分子在高亲和力位点的相互作用调节oriC DNA构象。
During assembly of the E.coli pre-replicative complex (pre-RC), initiator DnaA oligomers are nucleated from three widely separated high-affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low-affinity DnaA recognition sites, but is also regulated by a switch-like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high-affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high-affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin-selective recombineering methods to mutate E.coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high-affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high-affinity sites regulates oriC DNA conformation.