Haykinson M J, Johnson L M, Soong J, Johnson R C
Department of Biological Chemistry, UCLA School of Medicine 90095-1737, USA.
Curr Biol. 1996 Feb 1;6(2):163-77. doi: 10.1016/s0960-9822(02)00449-9.
Hin is a member of an extended family of site-specific recombinases--the DNA invertase/resolvase family--that catalyze inversion or deletion of DNA. DNA inversion by Hin occurs between two recombination sites and requires the regulatory protein Fis, which associates with a cis-acting recombinational enhancer sequence. Hin recombinase dimers bind to the two recombination sites and assemble onto the Fis-bound enhancer to generate an invertasome structure, at which time they become competent to catalyze DNA cleavage and strand exchange. In this report, we investigate the role of the Hin dimer interface in the activation of its catalytic functions.
We show that the Hin dimer is formed at an interface that contains putative amphipathic alpha-helices in a manner that is very similar to gamma delta resolvase. Certain detergents weakened cooperative interactions between the subunits of the Hin dimer and dramatically increased the rate of the first chemical step of the reaction--double-strand cleavage events at the center of the recombination sites. Amino-acid substitutions within the dimer interface led to profound changes in the catalytic properties of the recombinase. Nearly all mutations strongly affected the ability of the dimer to cleave DNA and most abolished DNA strand exchange in vitro. Some amino-acid substitutions altered the concerted nature of the DNA cleavage events within both recombination sites, and two mutations resulted in cleavage activity that was independent of Fis activation in vitro. Disulfide-linked Hin dimers were catalytically inactive; however, subsequent to the addition of the Fis-bound enhancer sequence, catalytic activity was no longer affected by the presence of oxidizing agents.
The combined results demonstrate that the Hin dimer interface is of critical importance for the activation of catalysis and imply that interactions with the Fis-bound enhancer may trigger a conformational adjustment within the region that is important for concerted DNA cleavage within both recombination sites, and possibly for the subsequent exchange of DNA strands.
Hin是位点特异性重组酶大家族（DNA转化酶/解离酶家族）的成员之一，该家族催化DNA的倒位或缺失。Hin介导的DNA倒位发生在两个重组位点之间，需要调控蛋白Fis，它与顺式作用重组增强子序列结合。Hin重组酶二聚体与两个重组位点结合，并组装到与Fis结合的增强子上，形成一个转化体结构，此时它们能够催化DNA切割和链交换。在本报告中，我们研究了Hin二聚体界面在其催化功能激活中的作用。
我们发现Hin二聚体在一个含有假定两亲性α螺旋的界面处形成，其方式与γδ解离酶非常相似。某些去污剂削弱了Hin二聚体亚基之间的协同相互作用，并显著提高了反应第一步化学过程的速率——重组位点中心的双链切割事件。二聚体界面内的氨基酸取代导致重组酶催化特性发生深刻变化。几乎所有突变都强烈影响二聚体切割DNA的能力，并且大多数在体外消除了DNA链交换。一些氨基酸取代改变了两个重组位点内DNA切割事件的协同性质，两个突变导致切割活性在体外独立于Fis激活。二硫键连接的Hin二聚体无催化活性；然而，在添加与Fis结合的增强子序列后，催化活性不再受氧化剂存在的影响。
综合结果表明，Hin二聚体界面对于催化激活至关重要，这意味着与Fis结合的增强子的相互作用可能触发对两个重组位点内协同DNA切割以及可能对随后DNA链交换都很重要的区域内的构象调整。
