Identification of DNA binding sites, assigning domain function and domain interactions in the catalytic cycle of reverse gyrase
Reverse gyrase is a thermophilic enzyme that catalyzes the ATP-dependent introduction of positive supercoils into DNA. Reverse gyrase consists of a helicase and a topoisomerase domain. Both domains interact with the DNA substrate. The cooperative action of the helicase and topoisomerase domain allows for the ATP-dependent positive supercoiling of DNA. To understand the molecular details of the cooperation of both domains in DNA binding and supercoiling, we aim to identify the contributions of DNA binding sites in both domains to binding of the DNA substrate. Furthermore, we probed the role of interactions between the helicase and topoisomerase domain for reverse gyrase activity.
Thermotoga maritima reverse gyrase contains two zinc fingers, one in the helicase and one in the topoisomerase domain. The two zinc fingers are connected by a salt bridge between His9 and Asp631. We investigated the role of this salt bridge on DNA binding and supercoiling activity. To dissect the role of individual DNA binding sites in the helicase and topoisomerase domains to binding of the DNA substrate, we performed chemical and UV crosslinking of reverse gyrase /DNA complexes. The crosslinking analysis then allows for the identification of individual protein-DNA contacts. These studies will reveal the hitherto unknown mode of DNA binding to reverse gyrase at different stages of the nucleotide cycle.
Speaker: Dr. Soltan Mohammadi K. Nafiseh, Institut für Physikalische Chemie, Westfälische Wilhelms-Universität Münster, Germany