DNA topoisomerases are enzymes present in all domains of life. They are responsible for maintaining the topological state of DNA in cells through supercoiling, relaxation, catenation, decatenation, knotting and unknotting. DNA gyrase is a unique topoisomerase, present mainly in bacteria that can introduce negative supercoils into DNA utilizing ATP hydrolysis. DNA gyrase plays crucial roles in removing positive supercoils and introduce negative supercoils in processes like replication and transcription making it a great target for antibiotics. During supercoiling activity, gyrase undergoes extensive conformational changes, which makes it challenging to study gyrase structurally, hence very few low resolution models of full-length enzyme have been obtained. Decades of research brought a good mechanistic understanding of supercoiling cycle, however, the structural equivalents of detected mechanistic states have not been obtained yet. Recent technological advancement in the field of cryo electron microscopy (cryoEM) made it possible to obtain high-resolution structural information of challenging proteins. Here, I present two cryoEM oligomeric complexes of open dimers of gyrase A missing C-terminal domains (GyrA-ΔCTD) with 44 base pair DNA. Structures were solved without prior knowledge of the complex structure to 4Å and 5Å resolution. Open GyrA dimers in one of the oligomeric complexes form two types of assemblies with DNA passing through the open DNA gate or positioned right above the gate prior to passage. These DNA orientations correspond to a T-segment DNA that needs to pass through the DNA gate in order to introduce a negative supercoil. Due to transient nature of this interaction, this is the first time T-segment DNA inside the open Gyr A gate has been shown. Observed conformations have been predicted before but never observed. Protein – DNA interaction interfaces are positively charged and conserved among many bacterial species highlighting the relevance of presented structures. In addition, this is the second time an open structure of GyrA dimer has been reported. Comparison of our structures with those published before point to a larger conformational plasticity of the open state than previously assumed. CryoEM has also been used to study the structure of a full–length gyrase. Optimization of complex formation for structural studies have been performed with linear and small circular DNA as gyrase substrates. Supercoiling activity of the enzyme has been confirmed for small circular DNA. Variety of ATPase domain substrates have also been tested. Once good conditions were found, initial negative stain EM and cryoEM datasets were collected. Obtained two dimensional image averages show different conformational states of the enzyme with circular DNA wrapped around and trapped inside gyrase. This is the first time gyrase with circular DNA as a substrate has been observed. This result proves that small circular DNA can be successfully used as a substrate for gyrase. Optimized conditions will serve as a starting point for future cryoEM studies of gyrase.

Creator

• Soczek, Katarzyna M

DOI

• https://doi.org/10.21985/n2-anc1-4p10

Subject

• Biophysics

Language

• en

Alternate Identifier

• etdadmin_upload_658816
• http://dissertations.umi.com/northwestern:14579

Keyword

• cryoEM
• topoisomerase
• DNA gyrase

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright