Interactions of the Escherichia coli primosomal PriB protein with the single-stranded DNA. Stoichiometries, intrinsic affinities, cooperativities, and base specificities.
Interactions of the Escherichia coli primosomal PriB protein with the single-stranded DNA. Stoichiometries, intrinsic affinities, cooperativities, and base specificities.
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大肠杆菌原始体 PriB 蛋白与单链 DNA 的相互作用。
DOI：
10.1016/j.jmb.2010.02.009
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发表时间：
2010
影响因子：
5.6
通讯作者：
Bujalowski,Wlodzimierz
中科院分区：
文献类型：
--
作者：
Szymanski,MichalR;Jezewska,MariaJ;Bujalowski,Wlodzimierz
大肠杆菌primosomal PriB蛋白与单链DNA的相互作用的定量分析是使用定量荧光滴定，光交联，和分析ultracentrugation技术。用一系列单链（ss）DNA寡聚体的乙烯基衍生物进行化学计量研究。使用大分子竞争滴定（MCT）方法研究了与未修饰核酸的相互作用。PriB二聚体-ssDNA复合物的总位点大小，即复合物中被PriB二聚体封闭的核苷酸的最大数目，为12± lnt。该蛋白具有单个DNA结合位点，其位于二聚体内的中心，并且具有功能上均质的结构。化学计量和光交联数据表明，只有一个单一的单体的PriB二聚体从事与核酸的相互作用。PriB与长寡聚体结合的分析是使用统计热力学模型进行的，该模型考虑了潜在结合位点的重叠和协同相互作用。PriB二聚体以强的正协同性结合ssDNA。内在亲和力和协同相互作用都伴随着净离子释放，阴离子参与离子交换过程。内在的结合过程是一个熵驱动的反应，强烈表明，DNA协会诱导蛋白质的大的构象变化。与高嘌呤寡聚体相比，PriB蛋白显示出对高嘧啶寡聚体的显著强烈偏好，其具有高约三个数量级的内在亲和力。PriB蛋白活性的这些结果的意义进行了讨论。
Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer–ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12±1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.