DNA cleavage by Type ISP Restriction-Modification enzymes is initially targeted to the 3'-5' strand.
DNA cleavage by Type ISP Restriction-Modification enzymes is initially targeted to the 3'-5' strand.
复制标题
DOI：
10.1093/nar/gks1210
复制
发表时间：
2013-01
影响因子：
14.9
通讯作者：
Szczelkun MD
中科院分区：
文献类型：
--
作者：
van Aelst K;Šišáková E;Szczelkun MD
两种易位型I型限制性修饰酶碰撞后产生双链DNA断裂的机制尚不完全清楚。在这里，我们证明了相关的Type ISP限制性修饰酶LlaGI和LlaBIII可以在聚合易位和碰撞后协同切割DNA。当其中一种酶是缺乏内切酶活性的突变蛋白时，相对于野生型酶的3 ‘ -5 ’链的DNA切割仍然发生，其动力学和碰撞位点与两种野生型酶之间的反应相同。野生型酶的DNA切割活性仍然由完全缺乏Mrr核酸酶结构域的蛋白质变体和不能易位的解旋酶突变体激活。然而，尽管存在完整的核酸酶结构域，解旋酶突变体不能切割DNA。野生型酶的切割不会被不相关的蛋白质障碍激活。我们认为，ISP型酶的核酸酶活性在与另一种ISP型酶碰撞后被激活，并且需要三磷酸腺苷结合/水解，但令人惊讶的是，不需要核酸酶结构域之间的相互作用。在最初的快速内切酶活性之后，额外的DNA切割事件随后发生得更慢，导致初始双链DNA断裂的进一步加工。
The mechanism by which a double-stranded DNA break is produced following collision of two translocating Type I Restriction–Modification enzymes is not fully understood. Here, we demonstrate that the related Type ISP Restriction–Modification enzymes LlaGI and LlaBIII can cooperate to cleave DNA following convergent translocation and collision. When one of these enzymes is a mutant protein that lacks endonuclease activity, DNA cleavage of the 3′-5′ strand relative to the wild-type enzyme still occurs, with the same kinetics and at the same collision loci as for a reaction between two wild-type enzymes. The DNA nicking activity of the wild-type enzyme is still activated by a protein variant entirely lacking the Mrr nuclease domain and by a helicase mutant that cannot translocate. However, the helicase mutant cannot cleave the DNA despite the presence of an intact nuclease domain. Cleavage by the wild-type enzyme is not activated by unrelated protein roadblocks. We suggest that the nuclease activity of the Type ISP enzymes is activated following collision with another Type ISP enzyme and requires adenosine triphosphate binding/hydrolysis but, surprisingly, does not require interaction between the nuclease domains. Following the initial rapid endonuclease activity, additional DNA cleavage events then occur more slowly, leading to further processing of the initial double-stranded DNA break.
登录
查看更多内容
影响因子：
14.9
作者：
Smith RM;Josephsen J;Szczelkun MD
通讯作者：
Szczelkun MD
影响因子：
5.6
作者：
Szczelkun, MD;Janscak, P;Halford, SE
通讯作者：
Halford, SE
影响因子：
14.9
作者：
Sasnauskas G;Kostiuk G;Tamulaitis G;Siksnys V
通讯作者：
Siksnys V
影响因子：
14.9
作者：
Šišáková E;van Aelst K;Diffin FM;Szczelkun MD
通讯作者：
Szczelkun MD
影响因子：
5.6
作者：
RIGGS, AD;SUZUKI, H;BOURGEOIS, S
通讯作者：
BOURGEOIS, S