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.
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