RecA filament sliding on DNA facilitates homology search.
RecA filament sliding on DNA facilitates homology search.
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作者：
Ragunathan K;Liu C;Ha T
在同源重组期间，RecA在单链（ss）DNA上形成螺旋丝，其搜索同源双链（ds）DNA并催化互补碱基对的交换以形成新的异源双链体。使用具有高时空分辨率的单分子荧光成像工具，我们表征了RecA细丝和dsDNA之间的相遇复合物。我们提出的证据支持“滑动模型”，其中RecA丝扩散沿着dsDNA轨道。我们进一步表明，同源性可以检测滑动过程中。滑动发生时的扩散系数约为8000 bp 2/s，允许细丝在解离前取样数百个碱基对。模型表明，滑动可以加速同源性搜索多达200倍。同源识别可以发生在少至6个核苷酸的互补碱基对上，识别效率随着互补性的提高而提高。我们的数据代表了DNA结合的多蛋白质复合物的第一个例子，该复合物可以沿着另一个DNA沿着滑动以促进靶搜索。DOI：http://dx.doi.org/10.7554/eLife.00067.001细胞中的DNA分子不断受到辐射、化学物质和其他物质的轰击，在细胞分裂过程开始之前，细胞修复这些物质造成的损伤是很重要的。大多数DNA分子由两条DNA单链组成，它们通过氢键结合在一起，形成熟悉的双螺旋结构。在DNA分子容易受到的各种类型的损伤中，双链断裂是最危险的，因为如果不修复它们可能导致癌症。DNA分子使用四种碱基腺嘌呤、胞嘧啶、鸟嘌呤和胸腺嘧啶来储存遗传信息。在单链DNA中，这些碱基连接到由交替的糖和磷酸基团组成的主链上。双链DNA的一个重要特征是两条链中的碱基序列是互补的--腺嘌呤总是与胸腺嘧啶配对，胞嘧啶总是与鸟嘌呤配对。然而，将碱基对结合在一起的氢键非常弱，这意味着双螺旋的两条链可以很容易地被拉开。这些键的形成和断裂的容易程度对许多遗传过程至关重要。修复双链断裂的一种方法是用另一个DNA分子的未受损片段替换受损的DNA片段。这种交换DNA分子的过程被称为链交换，由能够同时与两个DNA分子相互作用的蛋白质催化。在这个过程中，重要的第一步是确定可用于修复断裂的DNA片段。Ragunathan等人现在报道了来自大肠杆菌实验的证据，支持一种模型，其中蛋白质催化剂（大肠杆菌中的RecA）大肠杆菌）与单链DNA结合形成丝状DNA-蛋白质复合物（RecA丝），然后RecA丝可以沿着双链DNA分子滑动以寻找碱基对的互补序列。高分辨率荧光成像显示，RecA细丝能够在细丝与DNA解离并在不同位置重新结合之前对数百个碱基对进行采样。这种滑动很大程度上是由RecA细丝和双链DNA之间的静电相互作用驱动的，细丝能够识别包含少至6个匹配碱基的匹配序列。Ragunathan等人估计，与不涉及滑动的机制相比，滑动在寻找匹配序列方面快了大约两个数量级，例如仅依赖于DNA分子和RecA细丝之间偶然相遇的模型。通过证明DNA-蛋白质复合物可以沿着另一个DNA分子滑动以寻找目标，这些结果可能会导致对其他系统的新见解，在这些系统中，蛋白质-核酸复合物必须定位特定的碱基序列。DOI：http://dx.doi.org/10.7554/eLife.00067.002网站
During homologous recombination, RecA forms a helical filament on a single stranded (ss) DNA that searches for a homologous double stranded (ds) DNA and catalyzes the exchange of complementary base pairs to form a new heteroduplex. Using single molecule fluorescence imaging tools with high spatiotemporal resolution we characterized the encounter complex between the RecA filament and dsDNA. We present evidence in support of the ‘sliding model’ wherein a RecA filament diffuses along a dsDNA track. We further show that homology can be detected during sliding. Sliding occurs with a diffusion coefficient of approximately 8000 bp2/s allowing the filament to sample several hundred base pairs before dissociation. Modeling suggests that sliding can accelerate homology search by as much as 200 fold. Homology recognition can occur for as few as 6 nt of complementary basepairs with the recognition efficiency increasing for higher complementarity. Our data represents the first example of a DNA bound multi-protein complex which can slide along another DNA to facilitate target search. DOI: http://dx.doi.org/10.7554/eLife.00067.001 The DNA molecules in cells are continuously bombarded with radiation, chemicals and other agents, and it is important for cells to repair the damage caused by these before the process of cell division begins. Most DNA molecules consist of two single strands of DNA that are held together by hydrogen bonds in the familiar double-helix structure. Of the various types of damage that DNA molecules are prone to, double-strand breaks are among the most dangerous because they can lead to cancer if they are not repaired. DNA molecules use four bases—adenine, cytosine, guanine, and thymine—to store genetic information. In single-stranded DNA these bases are attached to a backbone made of alternating sugar and phosphate groups. A crucial feature of double-stranded DNA is that the sequences of bases in the two strands are complementary to each other—adenine is always paired with thymine, and cytosine is always paired with guanine. However, the hydrogen bonds that hold the pairs of bases together are quite weak, which means that the two strands of the double helix can be pulled apart quite easily. The ease with which these bonds can be formed and broken is crucial for many genetic processes. One way to repair a double strand break is to replace the damaged stretch of DNA with an undamaged stretch from another DNA molecule. This process of swapping DNA molecules, which is called strand exchange, is catalyzed by a protein that is able to interact with two DNA molecules at the same time. An important first step within this process is identifying the stretch of DNA that can be used to repair the break. Ragunathan et al. now report evidence from experiments on Escherichia coli that support a model in which the protein catalyst (RecA in the case of E. coli) combines with a single strand of DNA to form a filamentous DNA–protein complex (RecA filament) that can then slide along a double-stranded DNA molecule to search for a complementary sequence of base pairs. High-resolution fluorescent imaging reveals that the RecA filament is able to sample several hundred base pairs before the filament dissociates from the DNA and rebinds at a different location. The sliding was largely driven by electrostatic interactions between the RecA filament and the double-stranded DNA, and the filament was capable of identifying matching sequences that contained as few as six matching bases. Ragunathan et al. estimate that sliding is about two orders of magnitude faster at finding matching sequences compared to mechanisms that do not involve sliding, such as models that rely solely on chance encounters between DNA molecules and the RecA filament. By showing that a DNA–protein complex can slide along another DNA molecule to search for a target, these results could lead to new insights into other systems in which it is necessary for protein-nucleic acid complexes to locate a particular sequence of bases. DOI: http://dx.doi.org/10.7554/eLife.00067.002