Facilitated Unbinding via Multivalency-Enabled Ternary Complexes: New Paradigm for Protein-DNA Interactions.
Facilitated Unbinding via Multivalency-Enabled Ternary Complexes: New Paradigm for Protein-DNA Interactions.
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DOI：
10.1021/acs.accounts.7b00541
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发表时间：
2018-04-17
影响因子：
18.3
通讯作者：
Chen P
中科院分区：
文献类型：
--
作者：
Chen TY;Cheng YS;Huang PS;Chen P
动态的蛋白质-DNA相互作用构成了高度稳健的细胞机器以实现细胞功能。大量的研究集中在DNA结合蛋白如何寻找和与其靶DNA片段相互作用，以及哪些细胞信号可以调节蛋白质结合，其中蛋白质浓度是最明显的一个。相反，如何通过蛋白质浓度调节蛋白质解结合已经回避了注意，因为蛋白质从DNA解结合通常是单分子反应，因此不依赖于浓度。最近来自多个研究小组的单分子研究发现，蛋白质浓度可以促进DNA结合蛋白的解结合，揭示了蛋白质解结合的调节是基因调控的另一种机制范例。在这个账户中，我们回顾了这些最近的体外和体内单分子实验，这些实验揭示了多种类型的DNA结合蛋白，包括序列非特异性DNA结合蛋白（例如，类核苷酸相关蛋白;即，NAP）、序列特异性DNA结合蛋白（例如，金属响应性转录调节因子CueR和ZntR），序列中性单链DNA结合蛋白（例如，复制蛋白A;即，RPA）和DNA聚合酶。对于体外实验，Marko的小组使用单分子磁镊荧光显微镜研究了在浓度增加的溶液中GFP标记的DNA结合的NAP与非标记的NAP的交换。较高的未标记的NAP浓度下的更快的荧光强度降低表明DNA结合的NAP与较高的游离NAP浓度进行更快的交换。Chen的研究小组使用单分子荧光共振能量转移测量来研究CueR从其同源寡聚DNA中的解结合。DNA结合态的平均微观停留时间随着周围CueR浓度的增加而变短，表明游离CueR蛋白可以通过辅助解离或直接取代促进DNA上现任蛋白的解结合。格林的小组使用全内反射荧光显微镜和DNA幕技术研究了RPA从单链DNA上的解结合。荧光强度-时间曲线显示，野生型RPA浓度越高，衰减越快，表明DNA结合的RPA在遇到过量的游离RPA时可以进行浓度促进的交换。货车Oijen的小组研究了噬菌体T7和E.大肠杆菌复制系统使用单分子荧光显微镜和DNA流拉伸试验的组合。当Y526F聚合酶突变体的浓度增加时，观察到持续合成能力有较大的下降，表明聚合酶的解结合也是浓度依赖性的。利用频闪成像和单分子跟踪，Chen的研究小组进一步推进了他们对活细菌细胞的研究。他们发现CueR及其同系物ZntR在体内显示出与各自DNA结合位点的浓度增强的解结合。从这些体外和体内单分子研究中已经出现了机制共识，这些研究涵盖了一系列具有不同生物学功能的蛋白质。它涉及蛋白质和DNA之间的多价接触。多价性使得能够形成作为中间体的三元复合物，其随后引起浓度增强的蛋白质解结合。由于多价接触在DNA相互作用蛋白中普遍存在，因此这种多价使能的易化解结合机制提供了调节蛋白质-DNA相互作用的潜在通用机制范例。
Dynamic protein-DNA interactions constitute highly robust cellular machineries to fulfill cellular functions. A vast amount of studies have focused on how DNA-binding proteins search for and interact with their target DNA segments and on what cellular cues can regulate protein binding, for which protein concentration is a most obvious one. In contrast, how protein unbinding could be regulated by protein concentration has evaded attention because protein unbinding from DNA is typically a unimolecular reaction and thus concentration independent. Recent single-molecule studies from multiple research groups have uncovered that protein concentration can facilitate the unbinding of DNA-bound proteins, revealing regulation of protein unbinding as another mechanistic paradigm for gene regulation. In this account, we review these recent in vitro and in vivo single-molecule experiments that uncovered the concentration-facilitated protein unbinding by multiple types of DNA-binding proteins, including sequence-nonspecific DNA-binding proteins (e.g., nucleoid-associated proteins; i.e., NAP), sequence-specific DNA-binding proteins (e.g., metal-responsive transcription regulators CueR and ZntR), sequence-neutral single-stranded DNA-binding proteins (e.g., Replication protein A; i.e., RPA), and DNA polymerases. For the in vitro experiments, Marko’s group investigated the exchange of GFP-tagged DNA-bound NAPs with non-tagged NAPs in the solution of increasing concentration using single-molecule magnetic-tweezers-fluorescence microscopy. The faster fluorescence intensity decrease with higher non-tagged NAP concentrations suggests that DNA-bound NAPs undergo faster exchange with higher free NAP concentrations. Chen’s group used single-molecule fluorescence-resonance energy-transfer measurements to study the unbinding of CueR from its cognate oligomeric DNA. The average microscopic dwell times of DNA-bound states become shorter with increasing CueR concentrations in the surrounding, demonstrating that free CueR proteins can facilitate the unbinding of the incumbent one on DNA through either assisted dissociation or direct substitution. Greene’s group studied the unbinding of RPAs from single-stranded DNA using total internal reflection fluorescence microscopy and DNA curtain techniques. The fluorescence-intensity-versus-time traces show faster decay with higher wild-type RPA concentrations, indicating that DNA-bound RPAs can undergo a concentration-facilitated exchange when encountering excess free RPA. van Oijen’s group investigated the leading/lagging-strand polymerase exchange events in the bacteriophage T7 and E. coli replication systems using a combination of single-molecule fluorescence microscopy and DNA-flow-stretching assay. The processivity was observed to have larger decrease when the concentration of the Y526F polymerase mutant increases, indicating that the unbinding of the polymerase is also concentration-dependent. Using stroboscopic imaging and single-molecule tracking, Chen’s group further advanced their study into living bacterial cells. They found CueR, as well as its homologue ZntR, shows concentration-enhanced unbinding from their respective DNA-binding sites in vivo. Mechanistic consensus has emerged from these in vitro and in vivo single-molecule studies that encompass a range of proteins with distinct biological functions. It involves multivalent contacts between protein and DNA. The multivalency enables the formation of ternary complexes as intermediates, which subsequently give rise to concentration-enhanced protein unbinding. As multivalent contacts are ubiquitous among DNA-interacting proteins, this multivalency-enabled facilitated unbinding mechanism thus provides a potentially general mechanistic paradigm in regulating protein-DNA interactions.
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影响因子：
56.9
作者：
Elf, Johan;Li, Gene-Wei;Xie, X. Sunney
通讯作者：
Xie, X. Sunney
影响因子：
3.7
作者：
Gibb B;Ye LF;Gergoudis SC;Kwon Y;Niu H;Sung P;Greene EC
通讯作者：
Greene EC
影响因子：
16.6
作者：
Chen TY;Santiago AG;Jung W;Krzemiński Ł;Yang F;Martell DJ;Helmann JD;Chen P
通讯作者：
Chen P
DOI：
10.1073/pnas.0805050105
发表时间：
2008-09-16
影响因子：
11.1
作者：
Doucleff, Michaeleen;Clore, G. Marius
通讯作者：
Clore, G. Marius
影响因子：
16
作者：
Duderstadt, Karl E.;Geertsema, Hylkje J.;van Oijen, Antoine M.
通讯作者：
van Oijen, Antoine M.