| Part I. The double helical nature of DNA, elegant as it is, makes its structure and metabolism much more complicated. A simple winding solution is precluded to many of the topological problems that accompany the DNA transactions. DNA topoisomerases are the enzymes that accomplish these feats by either passing one strand of the DNA through a break in the opposing strand (type I subfamily) or by passing a region of duplex through a double-stranded gap generated in a second DNA (type II subfamily).; Part I of this thesis first presents the structural studies of the ATPase domain of human topoisomerase IIalpha (a type IIA topoisomerase). The ADP bound structure shows a much more open conformation than the AMPPNP bound one. Comparison of these structures revealed rigid-body movement of the structural modules within the ATPase domain, suggestive of the motions of a molecular gate. Four subsequent mutant structures have disclosed the energetics of the conformational change and shed light on what triggers movement directly following ATP hydrolysis. Attempts to obtain type IIA topoisomerase•DNA complex structures are also discussed.; Part II. The prototype for a transcription factor controlled by posttranslational modification is Escherichia coli Ada protein, a chemosensor that both repairs methylation damage in DNA and coordinates the resistance response to genotoxic methylating agents. Our work on the C38G mutant of the N-terminal domain of Ada (N-Ada) showed that it cannot repair methyl phosphotriester damage on its own. However, in the presence of methanethiol, which replaced the thiol of Cys38 as the nucleophile, the mutant regained the repair function, and with the same stereo selectivity on the substrate. Interestingly, this engineered system also became a real enzyme, rather than a sacrificial protein as the wild type is. We also measured the binding constants of several artificial N-Ada/DNA pairs. Consistent with the electrostatic switch hypothesis, tighter binding was observed regardless of the origin of the negative charge decrease at the interface. |
