Studies On The Mechanism Of Genetic Recombination

这个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转座的关键步骤是涉及Mu DNA序列末端和靶DNA的一对DNA裂解和链转移，这些反应产生分支DNA中间体。这两个化学反应步骤发生在被称为转座体的高阶蛋白质-DNA复合体中，转座体的核心由两个由MUA转座酶蛋白四聚体突触的Mu末端DNA片段组成。转座体的组装和活性受一系列辅因子的控制：一种称为IAS的增强子DNA序列元件，它重叠着Mu操纵子序列和与其结合的Mu阻遏物，Mub蛋白，E.Coli编码的Hu和IHF蛋白，ATP和Mg++。 我们已经证明，在转座体中，Mu末端的DNA裂解和随后的Mu DNA末端的链转移都是由与转座体中的伙伴Mu DNA末端结合的MUA单体催化的。转座体中的一个转座酶单体已被证明连续催化每个转座子末端的所有化学步骤。通过比较含有DNA底物的手性硫代磷酸的活性，我们可以监测底物DNA与转座酶活性部位之间的相互作用模式。这项研究表明，靶DNA链转移步骤的活性部位配置发生了显著变化，为靶DNA链转移步骤的明显不可逆性提供了机制解释，这对系统的生物学至关重要。用荧光标记的蛋白质和DNA研究了Mu转座复合体中的分子相互作用。已经开发了基于荧光的工具来检测转座酶-DNA结合、Mu-端配对、稳定的突触复合体形成和Mu-端DNA变形。 MUB ATPase控制着Mu DNA转座的每个早期步骤：它帮助转座体组装，参与靶DNA位点的选择，激活MUA转座酶进行链转移反应，并保护转座体不被ClpX伴侣蛋白过早分解，直到链转移完成，转座中间产物为宿主复制蛋白的DNA复制做好准备。反过来，MUb的功能状态受ATPase循环及其与MUA相互作用的控制。利用灵敏的荧光显微镜/CCD摄像系统在单分子水平上研究Mub-DNA复合体的结构和功能方面的技术和仪器已经被开发出来。使用GFP标记的MUB，在各种反应条件下监测了固定在玻片表面的DNA单分子上MUB聚合物的组装和拆解。我们了解到：MUB并不均匀地包裹DNA，相反，它形成了稳定结合的聚合物的谨慎斑块，夹杂着不稳定结合的团簇。依赖于ATP的MUB聚合物的组装首先涉及A/T富集区的随机成核事件，在A/T富集区有优先的Mu转位位点。MUB的解离优先发生在聚合物的末端，并与三磷酸腺苷的水解紧密相连。MUA四聚体加速了MUB与DNA的解离，这一过程依赖于DNA环介导的MUB聚合物和MUA四聚体的结合。 在我们对Mu转座体与靶DNA相互作用的研究过程中，我们发现Mu转座对碱基对错配的靶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的新方法来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定位致病突变以及家庭成员之间的遗传多态。该方法灵敏，可以检测所有的单核苷酸替换和短多核苷酸替换，并且很容易适应各种应用。

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. One transposase monomer within the transpososome has been shown to successively catalyze all the chemical steps at each transposon end. 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. This study suggested a significant change in the active site configuration for the target DNA strand transfer step, providing a mechanistic explanation for the apparent irreversibility of the target strand transfer step, which is essential for the biology of the system. 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 ClpX chaperon protein until strand transfer is completed and the transposition intermediate is ready for DNA replication by the host replication proteins. 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 by using a sensitive fluorescence microscope/CCD camera system. 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 stably bound polymers interspersed with less stably bound clusters. ATP-dependent assembly of MuB polymers involves stochastic nucleation event preferentially at A/T rich regions where preferred Mu transposition sites are located. 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. 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.