Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function.
Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function.
复制标题
DOI：
10.7554/elife.08646
复制
发表时间：
2015-09-18
期刊：
影响因子：
7.7
通讯作者：
Kowalczykowski SC
中科院分区：
文献类型：
--
作者：
Bell JC;Liu B;Kowalczykowski SC
大肠杆菌单链DNA结合蛋白（ssDNA binding protein，SSB）是大肠杆菌中一类重要的单链DNA结合蛋白。SSB结合ssDNA的可变足迹为30-70个核苷酸，反映了ssDNA围绕SSB四聚体的部分或完全包裹。我们直接成像单分子的SSB包被的ssDNA使用全内反射荧光（TIRF）显微镜和观察到的核蛋白复合物的分子内凝聚超过预期的基础上简单的包装过渡。我们进一步研究了这一意想不到的性质，单分子力谱使用磁镊。在有利于完全包裹的条件下，SSB参与长程可逆的分子内相互作用，导致SSB-ssDNA复合物的缩合。RecO和RecOR与SSB相互作用，使复合物进一步浓缩。我们的数据支持这样的想法，RecOR-和其他可能的SSB相互作用蛋白质的功能（S）部分改变远程，宏观相互作用之间或整个核蛋白复合物通过显微镜改变包装和桥接遥远的网站。http://dx.doi.org/10.7554/eLife.08646.001染色体由两股DNA组成，它们交织成螺旋状。在DNA被复制之前，这些链可以分离形成单链DNA，并用于细胞中的其他过程。如果双链DNA被有害的辐射或化学物质破坏，只有一条链可以复制，或者当受损的链在修复过程中被酶选择性降解时，也可以形成单链DNA。称为单链结合蛋白（或简称SSB）的蛋白质与单链DNA分子结合以保护它们。一个单链DNA分子包裹着一组四个SSB蛋白（称为四聚体）。DNA包裹SSB四聚体的程度取决于环境条件。例如，在高水平盐的存在下--这是细胞内的典型现象--单链DNA包裹住SSB的所有四个亚基。然而，在较低的盐水平下，DNA仅包裹SSB四聚体中的一些单元。一个叫做重组的过程可以修复DNA中的断裂。在这个过程中，含有单链DNA的断裂DNA分子可以与来自携带相同遗传序列的完整双链DNA分子的匹配（或互补）链配对。一种名为RecO的蛋白质在RecR蛋白质的帮助下帮助将两条互补的DNA链退火在一起。然而，对于RecR和RecO来说，要实现这一任务，它们需要与占据单链DNA的固有SSB蛋白一起工作。当SSB蛋白质碍事时，他们如何找到匹配的序列尚不清楚。贝尔等人使用TIRF显微镜和单分子力光谱技术直接观察了大肠杆菌中的SSB是如何从大肠杆菌中分离出来的。大肠杆菌与单链DNA分子结合并包覆。实验表明，当盐的水平增加时，被SSB蛋白包被的单链DNA变得紧凑，DNA分子的长度减少，这一过程被称为“分子内缩合”。Bell等人发现，由于与单链DNA的不同区域相关的两个SSB四聚体相互作用形成稳定的“八聚体”，因此发生了缩合。在RecO和RecR的存在下，单链DNA进一步压缩。Bell等人提出，这些重组蛋白充当支架，将单链DNA的远距离伴侣位点聚集在一起。这种浓缩允许细胞中相距很远的两个DNA序列更快地找到彼此。下一个挑战是了解如何识别单链DNA的匹配区域，以及是什么导致SSB移动以允许其他修复蛋白进入DNA。DOI：http://dx.doi.org/10.7554/eLife.08646.002网站
Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites. DOI: http://dx.doi.org/10.7554/eLife.08646.001 Chromosomes consist of two strands of DNA that are intertwined as a helix. These strands can peal apart to form single-stranded DNA before the DNA is copied and for other processes in cells. Single-stranded DNA can also form if double-stranded DNA is damaged by harmful radiation or chemicals so that only one strand can be copied or when the damaged strand is selectively degraded by enzymes during the course of repair. Proteins called single-stranded binding proteins (or SSBs for short) bind to single-stranded DNA molecules to protect them. A molecule of single-stranded DNA wraps around a group of four SSB proteins (known as a tetramer). The degree to which DNA is wrapped around the SSB tetramer depends on the environmental conditions. For example, in the presence of high levels of salt—which is typical inside cells – single-stranded DNA wraps around all four subunits of the SSB. However, at lower salt levels, the DNA only wraps around some of the units in the SSB tetramer. A process called recombination can repair breaks in DNA. During this process, a broken DNA molecule that contains single-stranded DNA can pair with a matching (or complementary) strand from an intact double-stranded DNA molecule that carries an identical genetic sequence. A protein called RecO helps to anneal two complementary DNA strands together with the help of the RecR protein. However, for RecR and RecO to achieve this task, they need to work together with the resident SSB proteins that occupy single-stranded DNA. How they find matching sequences when SSB proteins are in the way is not clear. Bell et al. used techniques called TIRF microscopy and single-molecule force spectroscopy to directly observe how SSB from the bacterium E. coli binds to and coats individual molecules of single-stranded DNA. The experiments show that when the levels of salt increase, single-stranded DNA that is coated with SSB proteins becomes compacted and the length of the DNA molecules decreases, a process referred to as ‘intramolecular condensation’. Bell et al. found that condensation occurred because two SSB tetramers that are associated with different regions of the single-stranded DNA interact to form stable ‘octamers’. In the presence of RecO and RecR, the single-stranded DNA compacted even further. Bell et al. propose that these recombination proteins act as a scaffold to bring together distant partner sites of single-stranded DNA. This condensation allows two DNA sequences that can be far apart in the cell to find one another more quickly. The next challenge is to understand how the matching regions of single-stranded DNA are identified, and what causes the SSBs to move to allow other repair proteins to gain access to the DNA. DOI: http://dx.doi.org/10.7554/eLife.08646.002
大肠杆菌的阳离子运输。 vi。 k交换。
影响因子：
3.8
作者：
Epstein, W;Schultz, S G
通讯作者：
Schultz, S G