Separating speed and ability to displace roadblocks during DNA translocation by FtsK.
Separating speed and ability to displace roadblocks during DNA translocation by FtsK.
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DOI：
10.1038/emboj.2010.29
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发表时间：
2010-04-21
期刊：
影响因子：
11.4
通讯作者：
Sherratt, David J.
中科院分区：
文献类型：
--
作者：
Crozat, Estelle;Meglio, Adrien;Allemand, Jean-Francois;Chivers, Claire E.;Howarth, Mark;Venien-Bryan, Catherine;Grainge, Ian;Sherratt, David J.
即使在强力作用下，FtsK 也能以 >5 kb/s 的速度定向转位 dsDNA。在体内，FtsK 在细菌分裂隔膜上的作用是完成染色体解链和分离的最后阶段所必需的。尽管易位酶结构已存在，但 ATP 水解与 DNA 易位耦合的机制尚不清楚。在这里，我们使用共价连接的易位酶亚基来深入了解 DNA 易位机制。野生型亚基的共价三聚体有效二聚化，形成具有高易位活性和激活 XerCD-dif 染色体解联能力的六聚体。中央亚基发生催化突变的共价三聚体形成了具有两个突变亚基的六聚体，具有强大的 ATP 酶活性。他们在单分子实验中表现出野生型易位速度，激活了易位依赖性染色体解链，但在沿着 DNA 易位期间，置换三链寡核苷酸或与生物素-DNA 连接的链霉亲和素的能力受损。这种易位速度和移动路障的能力的分离与顺序护送机制比随机、交接或协调机制更一致。
FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism. Covalent trimers of wild-type subunits dimerized efficiently to form hexamers with high translocation activity and an ability to activate XerCD-dif chromosome unlinking. Covalent trimers with a catalytic mutation in the central subunit formed hexamers with two mutated subunits that had robust ATPase activity. They showed wild-type translocation velocity in single-molecule experiments, activated translocation-dependent chromosome unlinking, but had an impaired ability to displace either a triplex oligonucleotide, or streptavidin linked to biotin-DNA, during translocation along DNA. This separation of translocation velocity and ability to displace roadblocks is more consistent with a sequential escort mechanism than stochastic, hand-off, or concerted mechanisms.
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