标题：Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：37
页码：E5133-E5141
DOI：10.1073/pnas.1510814112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThis study develops and exploits an expanded single-molecule fluorescence technique to understand the molecular mechanism of assembly, translocation, and disassembly of a hexameric DNA motor, FtsK, which functions ubiquitously in bacterial chromosome segregation. Assembly of single hexamers on DNA and their subsequent rapid translocation were directly assayed. FtsK hexamers dissociated soon after encountering and activating XerCD-dif recombination complexes. This work contrasts with previously published reports, which suggested that FtsK can reverse spontaneously during translocation, or upon encountering XerCD-dif. Furthermore, in some previous assays, the readout was DNA looping; here, we show that looping does not occur with single hexamer translocation. The technique used provides a blueprint for mechanistic real-time studies of individual protein-nucleic acid complexes. The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly ([~]0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for [~]0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK-XerCD-dif encounter activating only one round of recombination.
关键词：tethered fluorophore motion ; DNA translocation ; site-specific DNA recombination ; chromosome segregation ; single-molecule FRET
