Date of Award
Doctor of Philosophy
Bacteriophage Mu is one of the model systems to study DNA transposition. The availability of an in vitro soluble system greatly facilitates the dissection of the transposition mechanisms at the molecular levels. Several aspects of the in vitro mini-Mu DNA transposition have been dealt with in this thesis.;First, the flanking host DNA sequences on the strand cleavage step mediated by Mu transposase (Mu A protein) was investigated. We found that certain flanking host sequences could inhibit the strand cleavage step without affecting the earlier synapsis step. Furthermore, this cleavage defect could be overcome by the Mu B protein in the presence of ATP.;Second, the role of the 10 kDa C-terminal domain (domain III) of Mu transposase was studied. We showed that the cloned domain III was still functional in interacting with the Mu B protein in the absence of the 65 kDa N-terminal domain of Mu transposase. Deletion analysis revealed that the last 36 residues at the C-terminus of Mu transposase were involved in interacting with the Mu B protein. An intact C-terminus was required for efficient interactions between the Mu A and Mu B proteins.;Third, we mapped a novel non-specific DNA binding and nuclease activity in a 26 residue region (aa575-600) at N-terminus of domain III of Mu transposase. We showed that this region was required in both early synapsis step and the subsequent strand cleavage step. We argue that this 26 residue region might contact the Mu-host junctions in the transpososomes. Complementation studies further suggest that the active sites for the strand cleavage activity of Mu transposase are made up of amino acids in this 26 residue region of domain III on one transposase monomer and the conserved acidic residues (D, D35E) of domain II on a separate transposase monomer.
Wu, Zhenguo, "The In Vitro Studies On Phage Mu Transposase" (1995). Digitized Theses. 2585.