Name: iterative optimization of dna duplexes for crystallization of seqa-dna complexes
Uploaded: 2012-11-01T15:00:00
Description: Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

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）。

1。蛋白质纯化的柔性连接器连接的N-（低聚化）和C-末端（DNA结合）域的SeqA辅助识别由一至三个匝上的DNA分离的半甲基化的GATC重复。在这项研究中，我们使用了二聚体的变种SeqA（SeqAΔ（41-59）-A25R）的N-端结构域的点突变，防止进一步寡聚化和DNA结合的串联GATC重复分离完全由一个的缩短链接器限制一匝的DNA上（ 图1）2,8。 <ol><li>与质粒编码SeqA的T7启动子...

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	<li>Campbell, J. L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+oriC+and+the+dnaA+gene+promoter+are+sequestered+from+dam+methyltransferase+following+the+passage+of+the+chromosomal+replication+fork.">E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork.</a> Cell. 62, 967-979 (1990).</li><li>Guarn&#233;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+SeqA-N+filament:+implications+for+DNA+replication+and+chromosome+organization.">Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization.</a> Embo. J. 24, 1502-1511 (2005).</li><li>Brendler, T., Austin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+SeqA+protein+to+DNA+requires+interaction+between+two+or+more+complexes+bound+to+separate+hemimethylated+GATC+sequences.">Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.</a> Embo. J. 18, 2304-2310 (1999).</li><li>Jordan, S. R., Whitcombe, T. V., Berg, J. M., Pabo, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+variation+in+DNA+length+yields+highly+ordered+repressor-operator+cocrystals.">Systematic variation in DNA length yields highly ordered repressor-operator cocrystals.</a> Science. 230, 1383-1385 (1985).</li><li>Tan, S., Hunziker, Y., Pellegrini, L., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+the+yeast+MATalpha2/MCM1/DNA+ternary+complex:+general+methods+and+principles+for+protein/DNA+cocrystallization.">Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization.</a> J. Mol. Biol. 297, 947-959 (2000).</li><li>Rice, P. A., Yang, S., Mizuuchi, K., Nash, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+IHF-DNA+complex:+a+protein-induced+DNA+U-turn.">Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn.</a> Cell. 87, 1295-1306 (1996).</li><li>Yang, W., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+site-specific+recombinase+gamma+delta+resolvase+complexed+with+a+34+bp+cleavage+site.">Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site.</a> Cell. 82, 193-207 (1995).</li><li>Chung, Y. S., Brendler, T., Austin, S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+cooperative+binding+of+SeqA+to+a+tandem+GATC+repeat.">Structural insights into the cooperative binding of SeqA to a tandem GATC repeat.</a> Nucleic Acids Res. , (2009).</li><li>Anderson, J., Ptashne, M., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocrystals+of+the+DNA-binding+domain+of+phage+434+repressor+and+a+synthetic+phage+434+operator.">Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator.</a> Proc. Natl. Acad. Sci. U.S.A. 81, 1307-1311 (1984).</li><li>Timsit, Y., Moras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+self-fitting:+the+double+helix+directs+the+geometry+of+its+supramolecular+assembly.">DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.</a> Embo J. 13, 2737-2746 (1994).</li><li>Chung, Y. S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+analysis+of+SeqA+bound+to+a+pair+of+hemimethylated+GATC+sites.">Crystallization and preliminary X-ray diffraction analysis of SeqA bound to a pair of hemimethylated GATC sites.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 64, 567-571 (2008).</li><li>DeLano, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL Molecular Graphic Systems. , (2002).</li></ol>

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?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录＃ </td><td> 评论（可选） </td></tr><tr><td> TRIS </td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td>乙二胺四乙酸（EDTA） </td><td> Fisher Scientific则</td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrol（DTT） </td><td>生物技术有限责任公司</td><td> DB0058 </td><td></td></tr><tr><td>氯化钠</td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td>甘油</td><td>卡利登</td><td> 5350-1 </td><td></td></tr><tr><td>蔗糖</td><td> Sigma-Aldrich公司</td><td> S5016-500G </td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td>尿素</td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40％的29:1双/丙烯酰胺</td><td>生物技术有限责任公司</td><td> A0007-500ML </td><td>储存于4°C </td></tr><tr><td>硼酸</td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td>二甲苯蓝FF </td><td> Bio-Rad公司</td><td> 161-0423 </td><td></td></tr><tr><td>溴酚蓝</td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td>可调式双垂直凝胶体系</td><td> CBC科技公司公司</td><td> DASG-250 </td><td></td></tr><tr><td>指数结晶屏幕</td><td>汉普顿研究</td><td> HR2-144 </td><td>储存于4°C </td></tr><tr><td>精灵我结晶屏</td><td>翡翠生物系统公司</td><td> EBS-WIZ-1 </td><td>储存于4°C </td></tr><tr><td>精灵II结晶屏</td><td&gt;绿生物系统公司</td><td> EBS-WIZ-2 </td><td>储存于4°C </td></tr><tr><td>经典结晶画面</td><td> Qiagen公司</td><td> 130701 </td><td>储存于4°C </td></tr><tr><td> Intelliplate盘</td><td>艺术罗宾斯工具</td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td>解决方案 蛋白质纯化用缓冲液：100mM的TRIS pH值为8，2mM的EDTA，1mM DTT和5％甘油。 蛋白储存缓冲液：20mM的TRIS pH值为8，150mM的NaCl，5mM的DTT，0.5mM EDTA和5％甘油。 凝胶装载组合：20克蔗糖，25毫克的溴酚蓝，二甲苯青FF 25毫克，1毫升10％w / v的SDS，10毫升10×TBE缓冲液加入到第70毫升蒸压DDH 2 O中温和加热搅拌直到溶解蔗糖和调整最终体积为100毫升与蒸压DDH 2 O存放于4℃ 2X上样缓冲液：加入11克尿素凝胶上样混合至10毫升。热板搅拌，直到尿素溶解。分装在2 mL管和存储在4°C。 10X PAGE混合：混合420.4克尿素，100毫升10×TBE缓冲液（蒸压），250毫升40％在DDH 2 O 29:1的双/丙烯酰胺搅拌，直至完全溶解并调节音量，以1公升。在黑暗的瓶子存放于4℃ 10X TBE：溶解 108克 TRIS，55克硼酸和9.3克EDTA DDH 2 O在1升在室温下的高压灭菌器和存储。 洗脱缓冲液：稀8毫升DDH 2 O200毫升上的5M NaCl，2毫升1 M TRIS液pH7.5，0.4毫升的0.5M EDTA pH值为8在室温下的高压灭菌器和存储。 </td></tr></tbody></tabLE&gt;

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

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。

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

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&gt;大肠杆菌</emSeqA，复制起始的负调控因子。

迭代优化的DNA双链的SeqA-DNA复合物的结晶

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

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X射线晶体学的蛋白质结晶

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

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在油脂介孔膜蛋白的结晶

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We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

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Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

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