Studies On The Mechanism Of Genetic Recombination

该项目的目标是揭示基因重排的分子机制。以噬菌体Mu的转座反应为模型系统进行了研究。Mu转座的关键步骤是一对DNA切割和链转移，涉及Mu DNA序列的末端和靶DNA;这些反应产生分支的DNA中间体。这两个化学反应步骤发生在称为转座体的高级蛋白质-DNA复合物中，其核心由两个MuA转座酶蛋白四聚体突触的Mu末端DNA片段组成。转座体的组装及其活性受许多辅因子的控制：称为IAS的增强子型DNA序列元件，其与Mu操纵子序列和与其结合的Mu阻遏子重叠，MuB蛋白，E.大肠杆菌编码的HU和IHF蛋白、ATP和Mg++。 我们已经表明，Mu末端DNA切割和随后在一个Mu DNA末端的链转移都是由与转座体内的伴侣Mu DNA末端结合的MuA单体催化的。通过比较含有手性硫代磷酸酯的DNA底物的活性，我们可以在整个连续的反应步骤中监测底物DNA和转座酶活性位点之间的相互作用模式。这项研究的结果导致了一个机制模型，解释了DNA转座的连续反应步骤如何在转座体内发生。用荧光标记的蛋白质和DNA研究了Mu转座复合物中分子间的相互作用。已经开发了基于双链的工具用于测定转座酶-DNA结合、Mu-末端配对、稳定的突触复合物形成和Mu-末端DNA变形。 MuB ATP酶控制Mu DNA转座的每个早期步骤：它协助转座体组装，参与靶DNA位点选择，激活MuA转座酶进行链转移反应，并保护转座体免受伴侣蛋白过早分解，直到链转移完成，转座中间体准备好进行DNA复制。反过来，MuB的功能状态由ATP酶循环及其与MuA的相互作用控制。技术和仪器已经发展到在单分子水平上研究MuB-DNA复合物的结构和功能方面。使用GFP标记的MuB，在各种反应条件下监测固定在载玻片表面上的单分子DNA上的MuB聚合物的组装和拆卸。我们了解到：MuB不均匀地包覆DNA，相反，它在DNA上形成MuB聚合物的离散补丁，其组装由随机成核事件引发。MuB解离优先从聚合物的末端发生，并且与ATP水解紧密耦合。MuA四聚体在依赖于MuB聚合物和MuA四聚体的DNA环介导的缔合的过程中加速MuB从DNA的解离。为这项研究开发的实验方法目前正在扩展到其他反应机制的研究中，包括HIV DNA整合反应和细菌质粒DNA分离过程。 在研究Mu转座体与靶DNA相互作用的过程中，我们发现Mu转座体对碱基错配的靶DNA位点具有很强的特异性。基于这一发现，我们开发了一种名为MutMap的新方法，用于检测和定位基因突变。通过这种方法，我们能够很容易地检测和绘制致病突变以及家庭成员之间的遗传多态性。该方法是敏感的，检测所有的单核苷酸取代以及短的多个核苷酸取代，并且容易适用于各种应用。

The objective of this project is to uncover the molecular mechanisms of genetic rearrangements. The transposition reaction of bacteriophage Mu is studied as a model system. Critical steps in Mu transposition are a pair of DNA cleavages and strand transfers involving the ends of Mu DNA sequence and a target DNA; these reactions generate a branched DNA intermediate. The two chemical reaction steps take place within higher order protein-DNA complexes called transpososomes, the core of which is composed of two Mu-end DNA segments synapsed by a tetramer of MuA transposase protein. Transpososome assembly and its activity is controlled by a number of cofactors: an enhancer type DNA sequence element called IAS that overlaps the Mu operator sequence and the Mu repressor that binds to it, the MuB protein, the E. coli-encoded HU and IHF proteins, ATP, and Mg++. We have shown that both the Mu end DNA cleavage and the subsequent strand transfer at one Mu DNA end are catalyzed by the MuA monomer that is bound to the partner Mu DNA end within a transpososome. By comparing the activity of chiral phosphorothioate containing DNA substrates, we could monitor the mode of interaction between the substrate DNA and the transposase active site throughout the successive reaction steps. The results of this study led to a mechanistic model that explained how the successive reaction steps of DNA transposition take place within a transpososome. The molecular interactions involved in Mu transposition complex have been studied by using fluorescence labeled proteins and DNA. Fluorescence-based tools have been developed for the assay of transposase-DNA binding, Mu-end pairing, stable synaptic complex formation, and Mu-end DNA deformation. MuB ATPase controls each of the early steps of Mu DNA transposition: it assists transpososome assembly, is involved in the target DNA site selection, activates the MuA transposase for strand transfer reaction, and protects transpososome from premature disassembly by a chaperon protein until strand transfer is completed and the transposition intermediate is ready for DNA replication. In turn, the functional state of MuB is controlled by the ATPase cycle and by its interaction with MuA. Techniques and instruments have been developed to study the structural and functional aspects of MuB-DNA complex at the single molecule level. Using GFP-tagged MuB, assembly and disassembly of MuB polymers on single molecules of DNA immobilized on a slide glass surface was monitored under a variety of reaction conditions. We learned that: MuB does not uniformly coat DNA, instead, it forms discreet patches of MuB polymers on the DNA, assembly of which is initiated by stochastic nucleation events. MuB dissociation takes place preferentially from the ends of a polymer and is tightly coupled to ATP hydrolysis. MuA tetramer accelerates dissociation of MuB from DNA in a process dependent on DNA-looping-mediated association of the MuB polymer and MuA tetramer. Experimental approach developed for this study is currently expanded in the study of other reaction mechanisms including the HIV DNA integration reaction and bacterial plasmid DNA segregation processes. In the course of our study on the Mu transpososome-target DNA interactions, we discovered that Mu transposition has a strong specificity to target DNA sites with base pair mismatches. Based on this finding, we developed a novel method called MutMap for detecting and mapping genetic mutations. With this method we were able to easily detect and map disease-causing mutations and also genetic polymorphisms among family members. The method is sensitive, detects all single nucleotide substitutions as well as short multiple nucleotide substitutions, and is easily adaptable for a variety of applications.