Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
Nat Commun. 2016 Aug 26;7:12568. doi: 10.1038/ncomms12568.
Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the 'proximal' and 'distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.
核苷酸切除修复（NER）可去除所有生命领域中化学性质不同的 DNA 损伤。在大肠杆菌中，UvrA 和 UvrB 启动 NER，尽管体内发生这种情况的机制细节仍有待确定。在这里，我们使用单分子荧光成像来全面描述活细胞中损伤搜索、识别和验证过程。我们表明，NER 起始涉及两步机制，其中 UvrA 扫描基因组并独立于 UvrB 定位 DNA 损伤。然后 UvrA 将 UvrB 从溶液中募集到损伤部位。这些步骤由“近端”和“远端”UvrA ATP 结合位点中的 ATP 结合和水解来协调。我们表明，最初的 UvrA 无 UvrB 依赖性损伤识别仅需要远端位点的 ATPase 活性。随后的 UvrB 募集需要近端位点的 ATP 水解。最后，UvrA 从损伤复合物中解离，允许 UvrB 协调下游的 NER 反应。
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