Wang Z, Namgoong S Y, Zhang X, Harshey R M
Department of Microbiology, University of Texas, Austin, Texas 78712, USA.
J Biol Chem. 1996 Apr 19;271(16):9619-26. doi: 10.1074/jbc.271.16.9619.
In an earlier kinetic study (Wang, Z., and Harshey, R. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 699-703), we showed that supercoiling free energy was utilized during Mu transposition to lower the activation barrier of some rate-limiting step in the formation of the cleaved Mu end synaptic complex (type I complex). We report here results from kinetic studies on the assembled but uncleaved synaptic complex (type 0). Based on the estimated rate constants for the formation of type 0 and type I complexes, as well as their temperature and superhelicity dependence, we infer that the type 0 complex is an authentic intermediate in the pathway to Mu end cleavage. Our results are consistent with type 0 production being the rate-limiting step in the overall type I reaction. The conversion of type 0 to type I complex is a fast reaction, does not show strong temperature dependence, and is apparently independent of substrate superhelicity. We have explored the DNA structure within the type 0 complex using chemical and enzymatic probes. The observed susceptibility of DNA outside the Mu ends to single-strand-specific reagents suggests that a helix opening event is associated with type 0 formation. This structural perturbation could account, at least partly, for the high activation barrier to the reaction. There is a close correlation between the appearance of single strandedness near the Mu ends and the superhelicity of the DNA substrate. It is possible that supercoiling energy is utilized in effecting specific conformational transitions within DNA. We have found that Zn2+ and Co2+ ions, like Mg2+ and Mn2+ ions, can efficiently cleave the type 0 complex. However, unlike Mg2+ and Mn2+ ions, Zn2+ and Co2+ ions cannot support assembly of type 0. We discuss the implications of our findings for the mechanism of Mu transposition.
在早期的动力学研究中（Wang, Z.和Harshey, R. M.（1994年），《美国国家科学院院刊》91卷，699 - 703页），我们表明在Mu转座过程中，超螺旋自由能被用于降低形成裂解的Mu末端突触复合体（I型复合体）过程中某些限速步骤的活化能垒。我们在此报告关于组装好但未裂解的突触复合体（0型）的动力学研究结果。基于对0型和I型复合体形成的估计速率常数，以及它们对温度和超螺旋度的依赖性，我们推断0型复合体是Mu末端裂解途径中的一个真实中间体。我们的结果与0型复合体的产生是整个I型反应中的限速步骤这一观点一致。0型复合体向I型复合体的转化是一个快速反应，对温度依赖性不强，并且显然与底物超螺旋度无关。我们使用化学和酶学探针探索了0型复合体内的DNA结构。观察到Mu末端之外的DNA对单链特异性试剂敏感，这表明螺旋打开事件与0型复合体的形成相关。这种结构扰动可能至少部分地解释了该反应的高活化能垒。Mu末端附近单链状态的出现与DNA底物的超螺旋度之间存在密切相关性。有可能超螺旋能量被用于实现DNA内特定的构象转变。我们发现Zn2+和Co2+离子与Mg2+和Mn2+离子一样，能够有效地裂解0型复合体。然而，与Mg2+和Mn2+离子不同，Zn2+和Co2+离子不能支持0型复合体的组装。我们讨论了我们的发现对Mu转座机制的意义。
