Key processes in the cell, such as replication, transcription and repair, result in the

supercoiling of genomic DNA, which is resolved by molecular machines known as

topoisomerases. Type II DNA topoisomerases remodel DNA by passing one duplex DNA

strand through a transient break in a second duplex DNA strand. Type II topoisomerases

achieve DNA cleavage and religation with high fidelity by the coordination of multiple

domains. Achilles heel-like enzymes, which are indispensible to cells, topoisomerases are

targeted by numerous antibiotics and cancer therapeutics, necessitating a thorough

understanding of their structure and function.

Previously thought to be a uniform group of enzymes, a number of bacterial type

IIA topoisomerase paralogs have been identified with highly specialized functions but it

is not understood how each paralog recognizes and acts on unique DNA topologies. The

central hypothesis of this project is that the bacterial type IIA DNA binding domain,

termed the C-terminal domain (CTD), is principally responsible for substrate

identification and functional output of type IIA topoisomerases. I performed an array of

structural and biochemical studies on three unique bacterial type IIA topoisomerases to

understand how the CTD controls type IIA topoisomerase activity. The work presented in

this dissertation identifies novel type IIA topoisomerase evolutionary paths, characterizes

novel regulatory mechanisms, and introduces evidence that many characteristics refine

enzyme activity.