诱导位点特异性复制遇阻<i>电子。大肠杆菌</i>用荧光阻遏操作员系统
Summary
We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.
Abstract
障碍物存在于DNA，包括紧密结合的蛋白质和各种病变，会严重抑制细胞的复制机制的进程。一个复制体的失速会导致其分解从染色体，部分或全部，从而导致复制叉的崩溃。从这个崩溃的恢复是细胞准确完整的染色体复制，随后将一个必需品。所发生还原的DNA的叉，并允许复制。因此，当合拢时，细胞已经发展多种机制以高的保真度来完成。先前，在细菌中这些复制修复途径已经使用紫外线的伤害，其具有不被局限于一个已知站点的缺点进行了研究。这份手稿描述了利用荧光阻遏操作员系统（FROS）创建一个特定站点蛋白块，能够引起失速和复制的崩溃FO系统RKS在大肠杆菌 。协议的细节如何复制的状态可在单个活细胞使用荧光显微镜和DNA复制中间体被可视化可以通过2维琼脂糖凝胶电泳进行分析。复制体部件（ 例如 DnaBts）的温度敏感突变体可以被合并到系统以诱导复制叉的一个同步崩溃。此外，所涉及的这些过程的重组蛋白质和解旋酶的作用可以使用本系统内的遗传击倒进行研究。
Introduction
在DNA复制，在复制体面临上影响其进程中的DNA的障碍。包括病变和差距DNA损伤以及异常的结构可以防止复制体从出发1。最近，人们已经发现，结合到DNA的蛋白是障碍的最常见的来源，以复制叉级数2。以下用核蛋白块的复制体的遭遇的事件的知识先前已受限于无法在已知位置来诱导在活细胞中的染色体这样的块， 在体外分析已经增强了我们的动力学行为的理解当它满足核蛋白堵塞3，以及复制体本身4,5的机械细节的活性复制体。复制的修复当前的理解通常与紫外线的损伤剂进行研究，并在体内 6-8使用质粒DNA </s了>。虽然可参与DNA修复它遇到的体内核蛋白块通常从这些研究中理解后，是否有由于在复制块中的不同原因的修复途径中的分子事件的变化仍还蛋白待确定。
在这里，我们描述了一个系统，它允许在使用荧光阻遏算系统（FROS）的染色体的特定位置，以建立一个核蛋白块。我们利用大肠杆菌菌株已具有整合到染色体9 240 TETO位点的阵列的大肠杆菌 。阵列内的每个TETO站点有侧翼它通过防止在阵列内的RecA介导的重组，以增加阵列的稳定性有10 bp的随机序列。这个数组，它的变化，原本是用来理解E.大肠杆菌染色体动态10,11但随后适应preveNT 在体内的复制12。阵列已经发现稳定地保持并且当由四面体10,12结合到方框接近复制叉的100％。利用体外的类似LACO阵列已经发现少至22部位为足以阻挡90％的复制，尽管这更短的阵列是体内 13不太有效。为了适应阵列创建核蛋白堵塞，阻遏蛋白必须高度优化的条件下在那里再结合数组来创建一个路障下生产过剩。堵塞的形成，以及随后的释放，可以通过使用荧光显微镜如果使用四环素阻遏的荧光标记的变体进行监测。在每一个细胞复制的状态被看作灶的数字，其中一个焦点是指只有一个数组的副本是细胞内的存在，多灶指示复制活跃的指示。这再次活跃当核蛋白堵塞是通过添加减小四面体的操作员站点的结合亲和力充分的复制体通过阵列进行的无端诱导的逆转褶皱被启用。阻遏蛋白仍然能够结合具有足够高亲和力的DNA阵列的现在的多个拷贝可以被可视化。
在核蛋白堵塞的事件的更复杂的细节可以使用中性中性二维琼脂糖凝胶电泳和Southern杂交14-16被发现。这些技术使得DNA结构在人群的分析。那些在事件过程中形成的，并有可能在复制中间体保持未修理，可以显现。通过改变限制性内切酶和利用探头，中间体可不仅在阵列区域而且上游阵列的当复制叉退化17,18被可视化。该回归需要放置在复制体解离随后;前缘和滞后初生根丝从模板链和退火分离成彼此产生四通的DNA结构（霍利迪交界处）的模板链的同时重新退火。
使用该系统，已经表明，当它遇到该块18中的复制叉并不稳定。此外，可以利用复制体部件的温度敏感的衍生物，以防止一旦倒塌复制叉的再填装。一旦块被建立，该菌株可以转移到非许可温度下，以确保该复制体的同步失活和受控预防重装的。这种温度引起的失活可确保所有人口都折叠在给定时间内的拨叉，并允许当复制体倒塌，该DNA是如何处理的，和需要什么来重新发生什么评估启动DNA复制的过程。
这里描述的系统的一个优点是，核蛋白嵌段是完全可逆的;因此，该细胞的从核蛋白块中恢复的能力能够被遵循。加入无水四环素向细胞将缓解阻遏的紧密结合，使一个复制叉继续通过与细胞重新获得活力。堵塞的溢流可以通过中性中性二维琼脂糖凝胶电泳5分钟后进行可视化，并通过在10分钟内显微镜。此外，可行性分析可以揭示该菌株从复制堵塞恢复并继续增殖的能力。
通过改变在此处所述的实验过程中使用的菌株的遗传背景，对于这种类型的堵塞的修复途径可以阐明。
Protocol
Representative Results
Discussion
During chromosome duplication, the replication machinery will encounter various impediments that prevent its progress. To ensure the entire single-origin chromosome is replicated, bacteria have numerous pathways for repair of the DNA that then enables the replisome to be reloaded20,21. Lesions, single stranded breaks, double stranded breaks and proteins tightly bound to the DNA may each be dealt with using a dedicated pathway, although there is likely to be significant overlap in these pathways. The most commo…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Australian Research Council [DP11010246].
Materials
| Tryptone | Sigma-Aldrich | 16922 | Growth media component |
| Sodium Chloride | VWR | 27810.364 | Growth media component |
| Yeast extract | Sigma-Aldrich | 92144 | Growth media component |
| Potassium phosphate monobasic | Sigma-Aldrich | P9791 | Growth media component; Potassium buffer component |
| Potassium phosphate dibasic | Sigma-Aldrich | P3786 | Growth media component; Potassium buffer component |
| L-Arabinose | Sigma-Aldrich | A3256 | For induction of TetR-YFP production |
| Anhydrotetracycline hydrochloride | Sigma-Aldrich | 37919 | Release of replication bloackage |
| Axioskop 2 Fluorescence microscope | Zeiss | 452310 | Visualization of cells |
| eYFP filter set | Chroma Technology | 41028 | Visualization of YFP |
| CCD camera | Hamamatsu | Orca-AG | Visualization of cells |
| MetaMorph Software (Molecular Devices) | SDR Scientific | 31282 | Version 7.8.0.0 used in the preparation of this manuscript |
| Agarose | Bioline | BIO-41025 | For agarose plugs and gel electrophoresis |
| Original Glass Water Repellent (Rain-X) | Autobarn | DIO1470 | For agarose plug manufacture |
| TRIS | VWR | VWRC103157P | TE, TBE buffer component |
| Ethylene diaminetetraacetic acid | Ajax Finechem | AJA180 | 0.5 M EDTA disodium salt solution adjusted to pH 8.0 with NaOH. |
| Sodium azide | Sigma-Aldrich | S2002 | Bacteriostatic agent |
| Hydrochloric acid | Sigma-Aldrich | 258148 | TE buffer component |
| Sodium deoxycholate | Sigma-Aldrich | D6750 | Cell lysis buffer component |
| N-Lauroylsarcosine sodium salt (Sarkosyl) | Sigma-Aldrich | L5125 | Cell lysis and ESP buffer component |
| Rnase A | Sigma-Aldrich | R6513 | Cell lysis buffer component |
| Lysozyme | Amresco | 6300 | Cell lysis buffer component |
| Proteinase K | Amresco | AM0706 | ESP buffer component |
| Sub-Cell Model 192 Cell | BioRad | 1704507 | Electrophoresis system |
| UV transilluminator 2000 | BioRad | 1708110 | Visualization of DNA |
| Ethidium Bromide | BioRad | 1610433 | Visualization of DNA |
| Boric acid | VWR | PROL20185.360 | TBE component |
| Hybond-XL nylon memrbane | Amersham | RPN203S | Zeta-Probe Memrbane (BioRad 1620159) can also be used |
| 3MM Whatman chromatography paper | GE Healthcare Life Sciences | 3030690 | Southern blotting |
| HL-2000 Hybrilinker | UVP | 95-0031-01/02 | Crosslinking of DNA and hybridization |
| Deoxyribonucleic acid from salmon sperm | Sigma-Aldrich | 31149 | Hybridization buffer component |
| Sodium hydroxide | Sigma-Aldrich | S5881 | Denaturation buffer component |
| Trisodium citrate dihydrate | VWR | PROL27833.363 | Transfer buffer |
| Sodium dodecyl sulphate (SDS) | Amresco | 227 | Wash buffer component |
| Bovine serum albumin | Sigma-Aldrich | A7906 | Hybridization buffer component |
| Random Hexamer Primers | Bioline | BIO-38028 | |
| Klenow fragment | New England BioLabs | M0212L | |
| dNTP Set | Bioline | BIO-39025 | |
| Adenosine 5’-triphosphate-32P-ATP | PerkinElmer | BLU502A | |
| Storage Phosphor Screen | GE Healthcare Life Sciences | GEHE28-9564-76 | BAS-IP MS 3543 E multipurpose standard 35x43cm screen |
| Typhoon FLA 7000 | GE Healthcare Life Sciences | 28-9558-09 | Visualization of blot |
| Hybridization bottle | UVP | 07-0194-02 | 35 x 300mm |
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