Microsecond dynamics of protein-DNA interactions:: Direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer
Microsecond dynamics of protein-DNA interactions:: Direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer
复制标题
DOI：
10.1016/j.jmb.2006.02.070
复制
发表时间：
2006-05-26
影响因子：
5.6
通讯作者：
Ansari, Anjum
中科院分区：
文献类型：
--
作者：
Kuznetsov, Serguei V.;Kozlov, Alexander G.;Ansari, Anjum
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。同源四聚体蛋白的每个亚基都含有一个潜在的单链DNA结合位点，因此该蛋白可以以多种结合模式与单链DNA结合，其中之一是(SSB)(65)模式，其中一段65个核苷酸的单链DNA与四聚体的所有四个亚基相互作用并包裹其周围。之前使用寡脱氧核苷酸 (dT)(70) 对 (SSB)(65) 复合物形成的停流动力学研究无法解决 ssDNA 快速包裹在四聚体周围的初始结合步骤。在这里，我们报告了一项激光温度跳跃研究，其分辨率在 500 ns 至 4 ms 时间范围内，可直接检测这些 ssDNA 缠绕/解开步骤。观察到双相时间过程具有与浓度无关的快相，并且发生在数十微秒的时间尺度上，反映了 ssDNA 围绕 SSB 四聚体的缠绕/展开。对较慢结合相的分析，结合平衡结合和停流动力学研究，也为形成 (SSB)(65) 复合物的途径中先前未检测到的中间体提供了证据。 (c) 2006 Elsevier Ltd. 保留所有权利。
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.