迭代优化的DNA双链的SeqA-DNA复合物的结晶
Summary
蛋白质-DNA复合物的晶体结构，可以提供蛋白质的功能，机制，以及特定的相互作用的性质的洞察。这里，我们报告如何优化共结晶与双链DNA的长度，序列和两端<em>大肠杆菌</emSeqA，复制起始的负调控因子。
Abstract
大肠杆菌 SeqA是一个负调节DNA复制，以防止过 早封存半甲基化的GATC集群内再引发事件的起源，复制1。除了 原点，SeqA被发现在复制叉，它举办新复制的DNA到更高的有序结构。 SeqA联营公司只有微弱的单GATC序列，但它形成高亲和力DNA双链包含多个GATC网站的配合。功能和结构的最小单元SeqA一个二聚体，由此说明的要求中的至少两个的GATC序列与半甲基化DNA的3形成的高亲和性的络合物。此外，SeqA的体系结构，与低聚和由柔性连接器分离的DNA结合结构域，允许绑定GATC重复分离最多三螺旋匝。因此，在分子水平上理解的功能SeqA需要的结构模拟裂解SeqA绑定到多个GATC序列。在蛋白质-DNA结晶，DNA可以没有一个特殊的效果取决于蛋白质和DNA的相对大小和体系结构在包装上的相互作用。如果蛋白质的DNA或脚印大多数的DNA的比大时，晶体堆积主要是介导的蛋白质 – 蛋白质相互作用。相反，当该蛋白质是相同的尺寸或小于该DNA，或者它仅覆盖的一小部分的DNA，DNA-DNA和DNA-蛋白质相互作用支配晶体包装。因此，蛋白质-DNA复合物的结晶化需要的DNA长度为4的系统的筛选和DNA末端（钝或悬）5-7。在这份报告中，我们将介绍如何设计，优化，净化，结晶半甲基化DNA双链串联GATC重复复杂的二聚体的变种SeqA（SeqAΔ（41-59）-A25R），以获得合适的晶体结构的决心。
Protocol
Discussion
高分子X射线晶体学面临的最大挑战之一是获得衍射晶体。在蛋白质或蛋白质-DNA复合物的情况下，这一挑战，加剧了由于必须进行优化的附加变量。人们普遍相信，相邻的DNA分子，在更长的伪全双工的主要参数进行优化以提高关联的DNA的长度和存在的粘出挑。然而，我们已经表明，这些悬伸的性质及长度可以有额外的影响的蛋白质-DNA复合物的晶体堆积。虽然这个协议设计的合理设计的寡核苷酸为?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者希望帮助DNA纯化感谢PXRR的工作人员在布鲁克海文国家实验室NSLS（）数据收集过程中的协助和莫妮卡皮隆方面奔去。这项工作是由加拿大卫生研究院（67189澳门元）。
Materials
| Name of the reagent | Company | Catalogue # | Comments (optional) |
| TRIS | Bioshop | TRS003.5 | |
| Ethylenediaminetetraacetic acid (EDTA) | Fisher Scientific | E478-500 | |
| Dithiotheitrol (DTT) | Bio Basic Inc. | DB0058 | |
| NaCl | Bioshop | SOD002.10 | |
| Glycerol | Caledon | 5350-1 | |
| Sucrose | Sigma-Aldrich | S5016-500G | |
| Sodium dodecyl sulfate (SDS) | Bioshop | SDS001.500 | |
| Urea | Bioshop | URE001.5 | |
| 40% 29:1 Bis/acrylamide | Bio Basic Inc. | A0007-500ml | Store at 4 °C |
| Boric acid | EMD | BX0865-1 | |
| Xylene cyanol FF | Bio-Rad | 161-0423 | |
| Bromophenol Blue | Bioshop | BR0222 | |
| Dual Adjustable Vertical Gel System | C.B.C. Scientific Company Inc. | DASG-250 | |
| Index crystallization screen | Hampton Research | HR2-144 | Store at 4 °C |
| Wizard I crystallization screen | Emerald BioSystems | EBS-WIZ-1 | Store at 4 °C |
| Wizard II crystallization screen | Emerald BioSystems | EBS-WIZ-2 | Store at 4 °C |
| Classics crystallization screen | Qiagen | 130701 | Store at 4 °C |
| Intelliplate trays | Art Robbins Instruments | 102-0001-00 | |
Solutions Protein purification buffer: 100 mM TRIS pH 8, 2 mM EDTA, 2 mM DTT and 5% glycerol. Protein storage buffer: 20 mM TRIS pH 8, 150 mM NaCl, 5 mM DTT, 0.5 mM EDTA and 5% glycerol. Gel loading mix: Add 20 g of sucrose, 25 mg of bromophenol blue, 25 mg of xylene cyanol FF, 1 ml of 10% w/v SDS and 10 ml of 10X TBE to 70 ml of autoclaved ddH2O. Stir with mild heating until sucrose is dissolved and adjust the final volume to 100 ml with autoclaved ddH2O. Store at 4 °C. 2X loading buffer: Add 11 g of urea to 10 ml of gel loading mix. Stir on a hot plate until urea dissolves. Aliquot in 2 ml tubes and store at 4 °C. 10X PAGE mix: Mix 420.4 g of urea, 100 ml of 10X TBE (autoclaved), 250 ml of 40% 29:1 Bis/Acrylamide in ddH2O. Stir until totally dissolved and adjust volume to 1 liter. Store in dark bottles at 4 °C. 10X TBE: Dissolve 108 g of TRIS, 55 g of boric acid and 9.3 g of EDTA in 1 liter of ddH2O. Autoclave and store at room temperature. Elution buffer: Dilute 8 ml of 5 M NaCl, 2 ml of 1 M TRIS pH 7.5, 0.4 ml of 0.5 M EDTA pH 8 on 200 ml of ddH2O. Autoclave and store at room temperature. |
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