Binding and cleavage of nicked substrates by site-specific recombinases XerC and XerD
Binding and cleavage of nicked substrates by site-specific recombinases XerC and XerD
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DOI：
10.1006/jmbi.1996.0709
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发表时间：
1997-01-10
影响因子：
5.6
通讯作者：
Sherratt, DJ
中科院分区：
文献类型：
--
作者：
Blakely, GW;Davidson, AO;Sherratt, DJ
在Xer位点特异性重组中，两个相关的重组酶XerC和XerD在一个位点dif催化链切割和重新连接反应，以确保大肠杆菌细胞分裂过程中正常的染色体分离。我们使用有缺口的自杀底物来托盘化反应中间体，并表明XerC在切割dif的顶部链时效率较高，而XerD在切割dif的底部链时效率较低。重组酶介导的切割位置由6个碱基对分开，发生在靠近重组酶结合位点的不同中心区域的两端。在不需要分子间突触的反应中，XerC可以在缺乏其伙伴重组酶的情况下低效地切割dif的顶部链。dif底部链上的缺口允许两个结合在相邻结合位点上的XerC原蛋白之间的合作相互作用，这表明XerC和XerD中都存在一个保守的相互作用域。两个相同的重组酶原体之间的协同作用不会发生在非缺口的线性DNA上。两个XerD催化突变蛋白的乙基化干扰足迹表明，整合酶家族RHRY四聚体中的保守结构域II精氨酸可能与可剪切磷酸盐直接接触。(C) 1997学术出版社有限公司
In Xer site-specific recombination two related recombinases, XerC and XerD, catalyse strand cleavage and rejoining reactions at a site, dif, in order to ensure normal chromosome segregation during cell division in Escherichia coli. We have used nicked suicide substrates to tray reaction intermediates and show that XerC cleaves the top strand efficiently while XerD is less efficient at cleaving the bottom strand of dif. Recombinase-mediated cleavage positions are separated by six base pairs and occur at either end of the dif central region adjacent to the recombinase binding sites. XerC can cleave the top strand of dif inefficiently in the absence of its partner recombinase during a reaction that does not require intermolecular synapsis. The presence of a nick in the bottom strand of dif allows cooperative interactions between two XerC protomers bound to adjacent binding sites, suggesting that a conserved interaction domain is present in both XerC and XerD. Cooperativity between two identical recombinase protomers does not occur on un-nicked linear DNA. Ethylation interference footprinting of two XerD catalytic mutant proteins suggests that the conserved domain II arginine from the integrase family RHRY tetrad may make direct contact with the scissile phosphate. (C) 1997 Academic Press Limited