Multiprotein E. coli SSB-ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions.
Multiprotein E. coli SSB-ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions.
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DOI：
10.1093/nar/gkaa1267
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发表时间：
2021-02-22
影响因子：
14.9
通讯作者：
Williams MC
中科院分区：
文献类型：
--
作者：
Naufer MN;Morse M;Möller GB;McIsaac J;Rouzina I;Beuning PJ;Williams MC
大肠杆菌 SSB (EcSSB) 是一种模型单链 DNA (ssDNA) 结合蛋白，对于基因组维护至关重要。 EcSSB 形成同源四聚体，以多种构象包裹 ssDNA，以促进 DNA 复制和修复。在这里，我们测量了许多 EcSSB 蛋白与固定张力下的单个长 ssDNA 基质的结合和包裹。我们表明 EcSSB 以双相方式结合，其中初始包裹事件之后是解包裹事件，因为 ssDNA 结合蛋白密度超过临界饱和度，并且高游离蛋白浓度增加了 EcSSB 处于较少包裹构象的比例。通过增加底物张力、减少底物长度和蛋白质突变来破坏 EcSSB 包裹的稳定性，我们还直接观察到一种不稳定的结合但未包裹的状态，其中 ssDNA 的 ∼8 个核苷酸被单个结构域结合，这可以充当过渡状态，通过该过渡状态发生 EcSSB-ssDNA 复合物的快速重组。当 ssDNA 过度饱和时，刺激解离会迅速去除多余的 EcSSB，留下一系列稳定包裹的复合物。这些结果提供了一种机制，通过该机制，稳定结合和包裹的 EcSSB 四聚体可以快速从 ssDNA 中去除，以实现 DNA 维持和复制功能，同时仍然在各种蛋白质浓度下充分保护 ssDNA。
Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.
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