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 the 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. These 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 normally is controlled by a number of cofactors: an enhancer-type DNA sequence element called IAS, the MuB protein, the E. coli-encoded HU and IHF proteins, ATP, and Mg++. Structurally and functionally important protein-DNA interactions within the transpososome were analyzed by assembling it from short Mu end DNA fragments and MuA under permissive reaction conditions, bypassing the need for many of the cofactors normally required for the process. Both the Mu end DNA cleavage and the subsequent strand transfer at one Mu DNA end were shown to be catalyzed by the MuA monomers that were bound to the partner Mu DNA end within a transpososome; this explains why Mu DNA end synapsis is a prerequisite for the catalytic steps. The role of the IAS in the transpososome assembly is currently under investigation by making use of a reaction system in which a transpososome is assembled from short Mu end DNA fragments, stimulated by the presence of the IAS containing DNA fragment. Efforts are continuing toward solving the high-resolution structure of domains of MuA transposase. The structures of the central catalytic core domain and the N-terminal IAS binding domain have previously been solved by X-ray crystallography and NMR techniques respectively. Most recently, in collaboration with scientists in LCP/NIDDK, two independently folded DNA binding subdomains of MuA that bind to two halves of the 22bp consensus binding sequences at the Mu ends have been determined by NMR techniques, and their DNA binding properties characterized.
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