Date of Award
Doctor of Philosophy
Transpososomes are higher order nucleoprotein structures which mediate the Mu in vitro strand transfer reaction. The product of the strand cleavage reaction, the Type 1 transpososome, has been further characterized. This complex constrains the nicked ends of Mu through a myriad of protein-protein and protein-DNA interactions and precedes the strand transfer step. At least two proteins are required for the efficient formation of this intermediate, Mu A and E. coli HU. Immunoelectron microscopic studies have revealed that both A and HU localize to greater than 90% of the complexes; however, a tetrameric unit of Mu A, as seen by protein cross-linking, is solely responsible for maintaining the structural and functional integrity of the transpososome. This Mu A tetramer protects three of the six Mu A end-type binding sites from nuclease digestion as well as an additional 78 base pairs at each Mu end extending into the host sequences. An enhanced sensitivity to hydroxyl radical cleavage occurring on the continuous strand just beyond the Mu-host junction has led us to suggest that an altered DNA structure such as a sharp kink may exist at this position.;The affinity of HU for the Mu transpososome is some 100 times greater than for supercoiled DNA. To better understand the role of HU in higher order complex assembly, this high affinity sequence independent DNA binding was studied using an HU-nuclease generated from chemical modification. Probing of Type 1 complexes with the HU-nuclease revealed specific binding of HU at the Mu left end. This pattern is consistent with wrapping of the DNA around an HU core to generate a tight loop. Immunoelectron microscopy has revealed a second DNA site undetected by the HU-nuclease and which has so far defied attempts at precise mapping. HU binding to this site is unlikely to rely on contacts with the Mu A tetramer since extensive chemical modification of either Mu A or HU did not disrupt the efficient interaction of HU with the transpososome. Moreover, the functional analogues of HU, E. coli IHF and bovine HMG-1, were also shown to efficiently bind the transpososome core.
Lavoie, Brigitte Denise, "Structural Aspects Of The Mu Transpososome" (1994). Digitized Theses. 2343.