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稳定结合的MUA转座酶蛋白四聚体突触的Mu-末端DNA片段组成。转座体的组装由许多辅助因子控制：一个称为IAS的增强子类型的DNA序列元件，它重叠了Mu操纵子序列和与其结合的Mu阻遏器，Mub蛋白，大肠杆菌编码的Hu和IHF蛋白，ATP和MG。利用一个简化的转座体组装反应系统，我们证明了Mu端DNA的裂解和随后的Mu DNA末端的链转移都是由MUA单体催化的，该单体结合到转座体中的伙伴Mu DNA末端。对于Tn10和Mu的转座，转座体内的两个转座酶单体被证明催化了转座子两端的所有化学步骤。通过使用含有DNA底物的手性硫代磷酸盐，我们比较了两种转座反应中不同反应步骤的底物在转座酶活性部位的结合方向。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。目前正在利用各种物理和生化技术对Mub-DNA复合体的结构和功能进行研究。用荧光标记的蛋白质和DNA研究了噬菌体Mu转座复合体中的分子相互作用。已经开发出用于分析转座酶-DNA结合、Mu-端配对、稳定突触复合体形成和Mu-端DNA变形的工具。实时研究了导致转座体组装的过程，以及反应过程中复合体内的构象变化。与LCP/NIDDK和芝加哥大学的科学家合作，继续努力解决MUA转座酶结构域以及蛋白质-DNA复合体的高分辨率结构。

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 stably bound tetramer of MuA transposase protein. Transpososome assembly 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++. By making use of a simplified transpososome assembly reaction system, 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. For Tn10 and Mu transposition, two transposase monomers within the transpososome have been shown to catalyze all the chemical steps at the two transposon ends. By using chiral phosphorothioate containing DNA substrates, we compared the orientation of the substrate engagement at the transposase active site for the different reaction steps in both transposition reactions. 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. Structural and functional aspects of MuB-DNA complex are currently under investigation by using a variety of physical and biochemical techniques. The molecular interactions involved in the transposition complex of phage Mu were studied by using fluorescence labeled proteins and DNA. Tools have been developed for the assay of transposase-DNA binding, Mu-end pairing, stable synaptic complex formation, and Mu-end DNA deformation. The processes that lead to the transpososome assembly, and also the conformational changes within the complex during the reaction are studied in real time. Efforts are continued toward solving the high-resolution structure of domains of MuA transposase as well as protein-DNA complexes in collaboration with scientists in LCP/NIDDK and at Chicago University.