Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus.
Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus.
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DOI：
10.1016/j.bios.2014.06.011
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发表时间：
2014-11-15
影响因子：
12.6
通讯作者：
Soultanas, Panos
中科院分区：
文献类型：
--
作者：
Green, Matthew;Gilhooly, Neville S.;Abedeen, Shahriar;Scott, David J.;Dillingham, Mark S.;Soultanas, Panos
单链DNA结合蛋白（单链DNA binding protein， SSB）是一种广泛存在的细菌必需蛋白，几乎参与了DNA代谢的各个方面。利用枯草芽孢杆菌SSB制备了一种无试剂SSB生物传感器，可作为枯草芽孢杆菌及其密切相关的革兰氏阳性菌解旋酶探针。我们已经证明了探针在使用来自芽孢杆菌的解旋酶的DNA解绕反应中的实用性，并首次表征了枯草芽孢杆菌SSB的DNA结合模式切换和化学计量学。SSB在DNA代谢中的重要性并不像之前认为的那样，仅仅局限于DNA复制过程中结合和保护ssDNA。它与一系列伙伴蛋白相互作用，协调DNA代谢的许多不同方面。在大多数情况下，它与伴侣蛋白的相互作用是物种特异性的，因此，了解如何在不同细菌中生产和使用同源的无试剂SSB生物传感器至关重要。在这里，我们解释了如何生产枯草芽孢杆菌SSB探针，该探针在与单链DNA结合时显示出9倍的荧光增强，并且可以用于所有相关的革兰氏阳性厚壁菌，这些厚壁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。生产枯草芽孢杆菌SSB探针的材料是市售的，因此这里描述的方法与以前发表的大肠杆菌SSB方法不同，可以广泛使用。已研制出枯草芽孢杆菌特异性生物传感器。由内部半胱氨酸残基引起的问题通过位点特异性诱变得以克服。FDA5M已商品化，可提高枯草芽孢杆菌生物传感器的生产。枯草芽孢杆菌SSB表现出与大肠杆菌SSB类似的盐依赖性dna结合模式切换。枯草芽孢杆菌SSB生物传感器可用于同源反应和相关厚壁菌的检测。
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. Bacillus subtilis specific biosensor has been produced. Problem posed by an internal cysteine residue was overcome by site-specific mutagenesis. FDA5M is commercially available enhancing the production of B. subtilis biosensor.. B. subtilis SSB exhibits salt dependent DNA-binding mode switch like E. coli SSB. The B. subtilis SSB biosensor can be used in cognate reactions and related firmicutes.
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影响因子：
2.9
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通讯作者：
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