The objective of this research is to understand the biochemical mechanism by which the Tn3 transposon causes insertions and other genetic rearrangements. The immediate goal is to characterize the activities and mechanisms of the Tn3 resolvase because these are the two Tn3-encoded proteins needed for transposition. We have found that the transposase is a site- specific DNA binding protein that binds to the inverted repeats at the ends of Tn3. We will determine the extent of binding by transposase to the inverted repeats down to the nucleotide level, and will also test altered inverted repeats. Transposase also has a DNA-dependent ATPase and we will optimize the conditions for this activity and search for other activities expected for a transposase protein. Establishment of an in vitro system for transposition will also be attempted. For resolvase, the mode of action of a series of chemical inhibitors will be investigated. Several of these inhibitors do not block DNA binding but do block DNA cleavage and recombination and we will test if they block the formation or maturation of a synaptic complex. We will also test the effect of these inhibitors for the gin inversion system in vitro. Inhibitors of DNA binding by resolvase will be testing for their effect on proteolytic fragments of resolvase. Along with isolation of resistant mutants, this should allow the identification of the domains of resolvase involved in DNA binding.
| Flanagan, P M; Fennewald, M A (1989) Analysis of inhibitors of the site-specific recombination reaction mediated by Tn3 resolvase. J Mol Biol 206:295-304 |
| Flanagan, P M; Finn, M C; Fennewald, M A (1989) Inhibitor analysis of synapsis by resolvase. J Biol Chem 264:16892-6 |
| Saldanha, R; Flanagan, P; Fennewald, M (1987) Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites. J Mol Biol 196:505-16 |
