Identification of Berenil Target Sites in Plasmid pBR322.
Identification of Berenil Target Sites in Plasmid pBR322.
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DOI：
10.19070/2332-2756-170004
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Liu S
中科院分区：
文献类型：
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作者：
Valenzuela MS;Green N;Liu S
Berenil是一种小沟DNA结合分子，已广泛应用于兽医学。建模研究表明，Berenil与DNA上富含A/T的区域结合，这种相互作用的产物导致相反DNA链之间形成交联。这些交联可能潜在地抑制基本的生物过程，包括转录和DNA复制。我们以前使用pBR 322基因组作为模型系统，研究A/T序列对Berenil活性的作用。我们报道了在pBR 322基因组中插入poly（dA）poly（dT）序列，当培养物暴露于Berenil时，会导致重组质粒的复制抑制。然而，我们注意到，即使在没有这些序列的情况下，亲本质粒复制也被抑制，尽管比重组体少。这一观察结果使我们进行了本研究，我们试图确定天然贝伦尼靶位点在pBR 322基因组中的位置。通过缺失分析、重组DNA和复制测定的组合，我们发现了一个378 bp的DNA片段，该片段具有Berenil靶位点的所有特征。缺乏该区域的重组质粒比亲本质粒对药物的折射率更高，而另一种含有该区域额外拷贝的变体增加了质粒对Berenil的易感性。378 bp的区域富含约60%的A/T，并含有约21个潜在的Berenil结合位点。
Berenil, a minor groove DNA binding molecule, has been extensively used in veterinary medicine. Modeling studies have suggested that berenil binds to A/T rich regions on the DNA and the product of this interaction causes the formation of crosslinks between opposite DNA strands. These crosslinks could potentially inhibit fundamental biological processes including transcription and DNA replication. We had previously used the pBR322 genome as a model system to investigate the role of A/T sequences on berenil activity. We reported that the insertion of poly(dA)poly(dT) sequences into the pBR322 genome causes replication inhibition of the recombinant plasmids when cultures were exposed to berenil. However, we noticed that even in the absence of these sequences the parental plasmid replication was also inhibited, albeit less than the recombinants. This observation led us to the present study were we attempted to identify the location of natural berenil target sites in the pBR322 genome. Through a combination of deletion analysis, recombinant DNA and a replication assay we uncovered a 378 bp DNA fragment that has all the hallmarks of a berenil target site. A recombinant plasmid lacking this region is more refractive to the drug than the parental plasmid, and another variant containing and extra copy of this region increases the susceptibility of the plasmid towards berenil. The 378 bp region is about 60% A/T rich and contains about 21 potential berenil binding sites.