Mfd Protein and Transcription-Repair Coupling in Escherichia coli.
Mfd Protein and Transcription-Repair Coupling in Escherichia coli.
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DOI：
10.1111/php.12675
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发表时间：
2017-01
影响因子：
3.3
通讯作者：
Selby CP
中科院分区：
文献类型：
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作者：
Selby CP
1989年，转录-修复偶联（TRC）首次在大肠杆菌中被描述，作为位于模板DNA链上的UV光产物的转录依赖性、优先核苷酸切除修复（NER）。这一发现导致了在Aziz Sancar教授的实验室中对TRC进行开创性的生化研究，当时，在了解UvrA， UvrB和UvrC蛋白在NER中的作用方面做出了重大贡献。当修复研究扩展到TRC时，发现模板而非编码链病变在体外阻断RNA聚合酶（RNAP），并且出乎意料的是，阻断的RNAP抑制了NER。一种转录修复偶联因子，也被称为Mfd蛋白，被发现可以去除被阻断的RNAP，将修复酶传递到病变部位，从而介导转录阻断病变部位比其他部位更快的修复。Mfd蛋白的结构和功能分析揭示了ATP水解和DNA结合的解旋酶基序，以及与RNAP和UvrA相互作用的区域。这些和其他的研究为其他研究者提供了基础，在接下来的几十年里，他们描述了Mfd令人着迷的和意想不到的结构和机制特征，揭示了TRC可能存在的其他途径，并发现了Mfd在细胞中的其他作用。转录-修复耦合中的关键中间体。RNA聚合酶（RNAP）通过Mfd蛋白与DNA连接，DNA部分包裹在Mfd蛋白上。这种结构是在RNAP被模板链损伤阻断后形成的（这里是环丁烷胸腺嘧啶二聚体，T<>T）。Mfd从DNA中去除被阻断的RNAP和RNA。由此产生的束缚中间体中的Mfd具有与UvrA-UvrB核苷酸切除修复蛋白强烈结合的构象。下一步，结合修复蛋白并将其运送到受损部位，破坏Mfd的稳定，使其与DNA分离。最终的结果是优先的，相对快速的修复转录阻断损伤。
In 1989, transcription-repair coupling (TRC) was first described in Escherichia coli, as the transcription-dependent, preferential nucleotide excision repair (NER) of UV photoproducts located in the template DNA strand. This finding led to pioneering biochemical studies of TRC in the laboratory of Professor Aziz Sancar, where, at the time, major contributions were being made toward understanding the roles of the UvrA, UvrB and UvrC proteins in NER. When the repair studies were extended to TRC, template but not coding strand lesions were found to block RNA polymerase (RNAP) in vitro, and unexpectedly, the blocked RNAP inhibited NER. A transcription-repair coupling factor, also called Mfd protein, was found to remove the blocked RNAP, deliver the repair enzyme to the lesion, and thereby mediate more rapid repair of the transcription-blocking lesion compared to lesions elsewhere. Structural and functional analyses of Mfd protein revealed helicase motifs responsible for ATP hydrolysis and DNA binding, and regions that interact with RNAP and UvrA. These and additional studies provided a basis upon which other investigators, in following decades, have characterized fascinating and unexpected structural and mechanistic features of Mfd, revealed the possible existence of additional pathways of TRC, and discovered additional roles of Mfd in the cell. Pivotal Intermediate in Transcription-Repair Coupling. RNA polymerase (RNAP) is tethered to DNA via Mfd protein, with DNA partially wrapped around Mfd. This structure forms after RNAP becomes blocked by a template strand lesion (here, a cyclobutane thymine dimer, T<>T). Mfd removes blocked RNAP and RNA from DNA. Mfd in this resulting tethered intermediate assumes a conformation that binds strongly to the UvrA-UvrB nucleotide excision repair proteins. The next step, binding to and delivering the repair proteins to the damage, destabilizes Mfd, which dissociates from DNA. The net result is preferential, relatively rapid repair of transcription-blocking damage.