Green Matthew, Gilhooly Neville S, Abedeen Shahriar, Scott David J, Dillingham Mark S, Soultanas Panos
School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
Biosens Bioelectron. 2014 Nov 15;61(100):579-86. doi: 10.1016/j.bios.2014.06.011. Epub 2014 Jun 11.
Single-stranded DNA-binding protein (SSB) is a well characterized ubiquitous and essential bacterial protein involved in almost all aspects of DNA metabolism. Using the Bacillus subtilis SSB we have generated a reagentless SSB biosensor that can be used as a helicase probe in B. subtilis and closely related gram positive bacteria. We have demonstrated the utility of the probe in a DNA unwinding reaction using a helicase from Bacillus and for the first time, characterized the B. subtilis SSB's DNA binding mode switching and stoichiometry. The importance of SSB in DNA metabolism is not limited to simply binding and protecting ssDNA during DNA replication, as previously thought. It interacts with an array of partner proteins to coordinate many different aspects of DNA metabolism. In most cases its interactions with partner proteins is species-specific and for this reason, knowing how to produce and use cognate reagentless SSB biosensors in different bacteria is critical. Here we explain how to produce a B. subtilis SSB probe that exhibits 9-fold fluorescence increase upon binding to single stranded DNA and can be used in all related gram positive firmicutes which employ drastically different DNA replication and repair systems than the widely studied Escherichia coli. The materials to produce the B. subtilis SSB probe are commercially available, so the methodology described here is widely available unlike previously published methods for the E. coli SSB.
单链DNA结合蛋白（SSB）是一种特征明确的普遍存在且必需的细菌蛋白，几乎参与DNA代谢的各个方面。利用枯草芽孢杆菌的SSB，我们构建了一种无需试剂的SSB生物传感器，可作为枯草芽孢杆菌及密切相关革兰氏阳性菌中的解旋酶探针。我们已证明该探针在使用枯草芽孢杆菌解旋酶的DNA解旋反应中的效用，并首次对枯草芽孢杆菌SSB的DNA结合模式转换和化学计量进行了表征。SSB在DNA代谢中的重要性并不局限于如之前所认为的那样，仅在DNA复制过程中简单地结合和保护单链DNA。它与一系列伴侣蛋白相互作用，以协调DNA代谢的许多不同方面。在大多数情况下，其与伴侣蛋白的相互作用具有物种特异性，因此，了解如何在不同细菌中产生和使用同源的无需试剂的SSB生物传感器至关重要。在此，我们解释如何制备一种枯草芽孢杆菌SSB探针，该探针在与单链DNA结合时荧光增强9倍，可用于所有相关的革兰氏阳性厚壁菌，这些细菌采用与广泛研究的大肠杆菌截然不同的DNA复制和修复系统。制备枯草芽孢杆菌SSB探针所需的材料可从商业渠道获得，因此与先前发表的大肠杆菌SSB方法不同，这里描述的方法广泛适用。
