Name: direct restart of a replication fork stalled by a head-on rna polymerase
Uploaded: 2010-04-29T17:00:00
Duration: 7 min 27 s
Description: Watch this Scientific Journal Video about Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase at JoVE.com

我们特别感谢赛斯达斯特提供RNAP和MFD蛋白表达载体。我们也感谢谢尔盖Borukhov GreB和张丹提供技术支持。这项工作是支持由来自（按付款当日价格计算）国家卫生研究院的资助，由玛丽 - Josee和亨利克拉维斯在洛克菲勒大学（RTP）的奖学金。

使用这种方法是在报告的研究<a href="http://www.sciencemag.org/cgi/content-nw/full/327/5965/590/F3">，科学327，590-592（2010）波默朗茨和奥唐奈</a> 。 1。大会暂停的RNA聚合酶伸长复杂如下： 500 nm的终浓度的大肠杆菌大肠杆菌 RNA聚合酶σ70，他是5纳米的生物素标记的3.6 kb的DNA模板含有100μL缓冲液（20毫米的Tris - CL（pH值7.5），MgCl 2的8毫米，0.5毫米EDTA，5 T7A1发起人的最终浓度混合MM数码地面电视，10％甘油）为10分钟，在37 ° C。 APU和40微米的GTP，ATP和CTP的100微米添加一个额外的10分钟在37 ° C 2。停止RNAP伸长复杂固定到链霉亲和素珠是执行如下： 200μL和素磁性包被的磁珠（Invitrogen公司），分别加入上述反应在室温为10分钟。 3。固定的停止RNAP伸长复杂的纯化步骤如下： 珠（从上面）洗净，用0.9缓冲液含0.75 M氯化钠，200微克/ ml肝素，和20μg/ ml的单链DNA（上清每个磁分离洗涤步骤后删除。）接下来，5倍珠洗2次，用0.9毫升缓冲液A 4。形成一个复制叉下游相对头对RNA聚合酶如下： 重悬于100μLNew England Biolabs公司缓冲区4和10个单位SAP我（New England Biolabs公司）珠（从上面）10分钟添加37 ° C。珠子用0.9毫升缓冲液A洗3次，然后重悬于50μL快速T4连接反应缓冲液（New England Biolabs公司）。 2μL快速T4连接酶（New England Biolabs公司）随着6纳米预退火分叉DNA终浓度在室温为10分钟（RP10，RP22，RP25）。珠洗3次，用0.9毫升缓冲液A 5。大会的复制体，并在复制叉的领先链合成的起始执行如下： DnaB（六聚体）的448 pmol珠150缓冲液孵育（20毫米的Tris - CL（pH值7.5），8 毫米氯化镁2，0.5毫米EDTA，5毫米的数码地面电视，10％甘油）30秒时23 ° C。 4.9 pmol *第三的波尔（波尔三，他减去β），15 pmolβ，ATP的2毫米，60微米dGTP和dATP的分别加入200μL的体积和进一步的5分钟，37℃孵育复制开始后，一起加入10微克SSB和24微米的dATP和dTTP [α- 32 P] dTTP的和[α- 32 P] dCTP（具体活动，3,000-5,000 CPM / pmol）终体积为250微升。反应终止后10分钟后，加入12μL500毫米EDTA。 6。的分离纯化和浓度的放射性标记的DNA产品如下： 珠（从上面），煮沸后终止反应，以消除珠DNA。删除任何剩余的DNA，其余珠，珠治疗的蛋白酶K，10 UL量为30分钟，在50 ° C。珠子，然后煮和磁分离上清液删除。合并后的上清液含有DNA纯化使用Qiagen公司PCR清理套件。纯净放射性标记的DNA产品，然后在碱性琼脂糖凝胶电泳分析。 *在头RNA聚合酶停止伸长复杂如上，但是，σ70的情况下复制被省略。 代表性的成果： 头部碰撞与复制体RNA聚合酶通常在两种产品的长度为2.5 kb和3.6 KB结果。 2.5 kb的产品，暂停从复制体RNA聚合酶和拖延后，碰撞与RNA聚合酶的叉长度的DNA。 3.6 kb的产品，要么不完整，入住的RNA聚合酶启动子或部分复制体的RNA聚合酶头通读全长度的DNA和结果。一个良好的或准确的结果表明，约50％的全长DNA产生。然而，在某些情况下会出现少占用由RNA聚合酶启动子和一个完整长度的DNA的比例更高，因为一个更大的replisomes人口是观察不遇到迎头的RNA聚合酶。复制的唯一完整长度的DNA（3.6 KB）在停止RNAP结果的情况下。 <img src="/files/ftp_upload/1919/1919fig1.jpg" alt="图1" /> 图。 1复制体是阻碍了头的RNA聚合酶 。 （一）实验装置。一个E。 COL我RNA聚合酶停止伸长复杂生物素标记的DNA包含如前所述T7A1发起人（9）组装。简单地说，一个停止伸长复杂所形成的孵化与生物素化DNA的RNA聚合酶全酶的APU，GTP，ATP和CTP此外还有10分钟的10分钟20 BPS下游发起人。链霉亲和素珠，添加额外的10分钟然后珠洗5次，每0.9毫升含0.75米，200 mg / ml的肝素，氯化钠和100 nm的单链DNA，以消除不稳定的RNA聚合酶- DNA复合物和多余的NTPS缓冲区。的DNA，然后与SAPI消化，水洗，结扎预退火分叉的DNA。珠被冲洗一遍之前的复制体大会。 （二）DnaB是那么的复制体组装和复制发起的存在（2巷）或缺乏（1巷）发起人是位于2.5 kb和前叉是面向对叉的直接转录。 RNA聚合酶。

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+DNA+repair:+two+decades+of+progress+and+surprises.">Transcription-coupled DNA repair: two decades of progress and surprises.</a> Science. 266, 1957-1957 (1994).</li><li>Selby, C. P., Sancar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+repair+and+human+disease.">Transcription-coupled repair and human disease.</a> J Biol Chem. 270, 4882-4882 (1995).</li><li>Liu, B., Alberts, B. 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洗涤停止伸长复杂与高盐的约束珠是至关重要的，以消除任何不稳定的非特异性结合的RNA聚合酶分子。 RNA聚合酶具有极高的亲和力引物 - 模板类型的DNA结构。因此，有必要消除这种非特异性的RNA聚合酶- DNA复合物，以执行复制的引物 - 模板。集中的放射性标记的DNA产品使用Qiagen公司的PCR纯化试剂盒也很关键，以获得足够高的放射性的信号，可以在凝胶观察。

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direct restart of a replication fork stalled by a head-on rna polymerase

体内研究表明，复制叉头转录复合物被逮捕到遇到。然而，命运的复制体和RNA聚合酶（RNAP）后，头部相撞是未知的。在这里，我们发现， 大肠杆菌碰撞后的大肠杆菌复制体摊位一个头转录复杂，而是倒塌，复制叉保持高度稳定和最终恢复后，取代了从DNA的RNA聚合酶的伸长率。我们还发现，转录修复偶联因子，MFD，促进直接重新启动的叉，下面的碰撞，促进RNA聚合酶的位移。这些研究结果表明复制设备的内在稳定性和新颖的作用在促进过去一个RNA聚合酶块复制的转录偶联修复途径。

Watch this Scientific Journal Video about Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase at JoVE.com

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

复制体的命运碰撞与头部的RNA聚合酶（RNAP）是未知的。我们发现，碰撞后的头对RNA聚合酶的复制体摊位，但恢复后，取代了从DNA的RNA聚合酶的伸长。 MFD促进便利碰撞后的RNA聚合酶的位移复制重新启动。

复制叉停滞了迎头RNA聚合酶直接重新启动

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诱导位点特异性复制遇阻<i&gt;电子。大肠杆菌</i&gt;用荧光阻遏操作员系统

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