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首页> 外文期刊>Journal of Molecular Biology >Microsecond dynamics of protein-DNA interactions: Direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer
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Microsecond dynamics of protein-DNA interactions: Direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer

机译：蛋白质-DNA相互作用的微秒动力学：直接观察大肠杆菌SSB四聚体周围单链DNA的包裹/解缠动力学

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The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)(65) mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)(65) complex formation using the oligodeoxynucleotide, (dT)(70), were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the similar to 500 ns to 4 ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)(65) complex. (c) 2006 Elsevier Ltd. All rights reserved.
机译：大肠杆菌单链DNA结合蛋白（SSB）在DNA复制，重组和修复过程中选择性地与单链（ss）DNA中间体结合。同型四聚体蛋白质的每个亚基都含有一个潜在的ssDNA结合位点，因此该蛋白质可以多种方式与ssDNA结合，其中一种是（SSB）（65）方式，其中ssDNA的65个核苷酸长的部分与并包裹四聚体的所有四个亚基。以前使用寡聚脱氧核苷酸（dT）（70）对（SSB）（65）复合物形成进行的停流动力学研究无法解决ssDNA在四聚体周围的快速包裹所引起的初始结合步骤。在这里，我们报告了一次激光温度跳跃研究，其分辨率大约在500 ns至4 ms的时间范围内，可直接检测这些ssDNA包装/展开步骤。观测到的双相时间过程具有与浓度无关的快速相，其发生时间为数十微秒，反映了ssDNA在SSB四聚体周围的包裹/解缠。较慢的结合相的分析，结合平衡结合和止流动力学研究，也为形成（SSB）（65）复合物的途径先前未被发现的中间体提供了证据。 （c）2006 Elsevier Ltd.保留所有权利。

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