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题名: 大肠杆菌同源重组修复机制研究
作者: 赵柏林
学位类别: 博士
答辩日期: 2015-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 汪海林
关键词: 毛细管电泳-激光诱导荧光偏振、RecA、ATP水解、RecA组装、 链交换,CE-LIFP, RecA protein, ATP hydrolysis, RecA assembly, strand exchange
其他题名: Study of E. coli RecA-mediated homologous recombination repair mechanisms
学位专业: 分析化学
中文摘要:       大肠杆菌RecA及家族蛋白在同源重组修复中发挥重要作用。然而RecA蛋白本身并不具有介导同源重组的功能,其需要在ATP条件下,在ssDNA上组装形成活性的RecA-ssDNA核蛋白复合物。RecA-ssDNA复合物具有ATP水解酶活性,同时其自身结构也受ATP水解调节而具动态特性。研究RecA蛋白在ssDNA上的组装,尤其是在生理ATP条件下的组装,进而寻找生理学相关的RecA组装形式有助于理解RecA介导同源重组的机制。此外,精准的同源重组有助于维持基因组的完整性,而错误倾向性的同源重组对生命体产生威胁。大肠杆菌体内同源重组具有非常高的准确性,而体外我们发现在ATPγS条件下形成RecA饱和的核蛋白复合物介导的同源重组准确性差。这也驱使我们寻找保证同源重组准确性的分子机制。
      我们首先将高效快速分离的毛细管电泳(CE)、高灵敏检测的激光诱导荧光(LIF)及分子大小敏感的荧光偏振(FP)结合发展了一种二维自由溶液分析方法,并用于研究RecA组装、组装复合物结构、组装复合物在同源重组链交换中的作用及其介导链交换准确性的机制。全文主要包括以下内容:
      1. CE-LIFP分析揭示无ATP条件下RecA在ssDNA上以高亲和力协同方式组装形成高组装复合物。无ATP条件下不同浓度RecA在TMR-T90上组装形成单一的复合物峰,且该峰具有相同的迁移时间及一致的大的荧光偏振响应,揭示RecA协同组装形成均一的高组装复合物。核酸酶酶切分析及无标记T90的竞争实验进一步确证了RecA的协同组装机制。
      2. 揭示RecA极度不饱和的低组装复合物是RecA生理学相关的组装形式。ATP水解抑制纳摩尔RecA在ssDNA上组装,但ATP水解刺激生理浓度(μM)的RecA组装形成高、中、低三种类型复合物,并且在生理ATP浓度条件下主要形成低组装复合物。利用短链的寡核苷酸探针建立RecA-ssDNA复合物结合计量学与其迁移时间的校正关系,并成功应用于计算不同组装度复合物的结合蛋白数,高、中、低三种类型复合物中每条ss83mer链分别结合24-27个、14-19个、5-7个RecA蛋白。中、低组装度复合物中有限的RecA数目表明大量的ssDNA区域没有RecA结合。复合物的荧光偏振及核酸酶酶切分析进一步验证了中、低组装度复合物的这种RecA不饱和的结构。
      3. 发现RecA极不饱和的低组装复合物是RecA介导链交换反应的活性形式。相对整体拉伸的RecA饱和的高组装复合物,低组装复合物更有利于链交换反应,并能保证链交换的准确性。RecA 430蛋白不形成高组装复合物但能够有效介导链交换反应,而无ATP水解能力的RecA K72R主要形成RecA饱和的复合物且不能介导链交换反应,这些进一步确证了低组装复合物在链交换中的重要作用。而RecA蛋白体内和体外一致的同源重组结果表明RecA蛋白在体内可能也通过低组装复合物介导链交换反应。
      4. 阐明RecA螺旋的间接解链活性是低组装复合物介导链交换反应的主要机制,并提出一种新颖的合理的同源重组模型---间接解旋模型。间接解旋模型指出低组装复合物通过碱基天然配对进行同源识别,这可能是RecA介导准确链交换的机制。
英文摘要:       E.coli RecA protein and its homologs play important roles in homologous recombination (HR). While RecA protein itself can not exert functions, it needs to assemble on ssDNA forming functional nucleofilaments in the presence of ATP. RecA-ssDNA filaments possess ATPase activity, however, ATP hydrolysis would regulate the dynamics of the filaments. The investigation on how does RecA assemble on ssDNA, especitally in physiologic conditions, and searching the physiology-related filaments will facilitate us to understand the mechanisms of homologous recombination (HR). Moreover, accurate HR help maintain genomic integrity, while error-prone HR will intimidate to organisms. The homologous recombination repair in E.coli keep higher fidelity. However, we found that the RecA-saturated nucleofilaments formed in the presence of ATPγS stimulate error-prone HR in vitro. All these phenomena drive us to unravel the mechanisms of accurate HR.
      we first developed a two-dimensional free solution assay using unique combination of highly efficient and fast capillary electrophoresis separation with ultrasensitive laser induced fluorescence and molecular size-sensitive fluorescence polarization. Then we investigated the assembly of RecA, the structures of RecA nucleofilaments, the roles of the filaments in medating HR and the mechanism of accurate strand exchange. The whole dissertation consists of contents as below:
      1. CE-LIFP analysis revealed that high affinity and cooperative assembly of RecA on ssDNA preferentially formed highly assembled filaments (HAFs) and was independent of ATP binding and hydrolysis. By assembly of different concentration of RecA on TMR-T90 in the absence of ATP, we found that only single type of complex formed, showing the same migration time shifts and fluorescne polarization reponses. This phenomenon indicated the cooperative assembly of RecA on ssDNA forming highly assembled filaments. Moreover, supernuclease digestaion analysis and competitive binding experiment with unlabeled T90 further confirmed the cooperative mechanism.
      2. We found that the unbound nucleotide sites-predominated RecA-ssDNA filaments, or lightly assembled filaments, are the physiology-related RecA filaments. ATP hydrolysis inhibits the assembly of nano-molar RecA on ssDNA,while the physiologic concentration of ATP (milli-molar) stimulates the formation of three types of filaments-lightly assembled (HAFs), moderately assembled (MAFs) and highly assembled filamnets (LAFs), with physiologic concentration of RecA (micro-molar). And the lightly assembled filaments are the physiology-related filaments. The estimated RecA bound numbers per ss83mer in LAFs, MAFs and HAFs were 5-7, 14-19 and 24-27, according to the correlation of RecA nucleofilaments migration shifts and bound RecA numbers per oligo. The limited RecA bound number in LAFs and MAFs indicated large amount of RecA-unbound sites exist in these filaments. The fluorescence polarizations and supernuclease digested assay further confirmed the unbound nucleotide sites-predominated structures of the filaments.
      3. We discovered that the LAFs facilitated strand exchange. Comparing to RecA-saturated and globally sretched nucleofilaments, the LAFs with predominantly unbound nucleotide sties mediated strand exchange with higher efficiency, moreover, it could promise the accuracy of strand exchange. We further showed that the RecA430 (G204S), which didn’t form highly assembled filaments, could mediate efficient strand exchange, but RecA K72R, which didn’t hydrolyze ATP and mainly formed RecA-saturated filaments, could not play roles in strand exhcnage. These results further confirmed the active roles of LAFs in medating strand exchange. The consistent phenomenon of homologous recombination of RecA mutants in vivo and in vitro indicated the active form of RecA participating in strand exchange in vivo might be also lightly assembled filaments.
      4. We found that the hlicace-like acitivty might be the mechanism of strand exchange mediated by LAFs. Further we proposed a new and rational homologous recombination model-indirect helicase model. This model indicated that homologous recognition was completed through bases’ natural anneling, which might contribute to the accuracy of strand exchange mediagted by LAFs.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34480
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
赵柏林. 大肠杆菌同源重组修复机制研究[D]. 北京. 中国科学院研究生院. 2015.
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