Topoisomerases are ubiquitous proteins found across all three domains of life (bacteria, archaea, and eukarya). They are involved in several cellular processes and the importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics. Topoisomerases change the topology of DNA by transiently breaking one (type I) or two (type II) DNA strands to allow passage of either a single or double DNA strand through the break or swiveling of one strand around the other. The study of the structure and function of topoisomerases promises not only to further our understanding of proteins that interact with DNA and alter its topological properties, but also to provide important information to aid in the design of new therapeutic agents. This proposal is concerned with biochemical, biophysical, and structural studies of different topoisomerases. The long term goal of the project is to provide a comprehensive understanding of topoisomerase action at many different length and time scales, from the atomic level to the nano scale. In the past period we made substantial progress towards this goal, including using single molecule methods to discover the role of pauses in the mechanism of type IA topoisomerases, characterizing the topoisomerase active site of topoisomerase V, solving the structures of 78 kDa and 97 kDa fragments of topoisomerase V containing one or two of the DNA repair sites, and discovering the presence of a third repair domain. In addition, we continue making progress on our work on a complex of gyrase with a large DNA fragment and also developed a new instrument that is capable of simultaneously manipulating single DNA molecules and reporting on movements of a protein by fluorescence. Our studies are providing important information on topoisomerases and allowing us to relate atomic structures to the wealth of existing functional, biochemical, and biophysical data. For the next project period we propose to continue and expand our studies of topoisomerases.
The specific aims for this proposal are: i) to probe the mechanism of type IA topoisomerases by novel single molecule approaches, ii) to continue our structural and biochemical studies of topoisomerase V, and iii) to continue our structural and mechanistic studies of a gyrase/DNA complex. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the macromolecules that we require for our work, X-ray crystallography and electron microscopy methods to solve their structures, and single molecule studies to elucidate their mechanism.

Public Health Relevance

We propose to study the structure and mechanism of DNA topoisomerases, enzymes that are involved in several cellular processes, such as transcription, replication and recombination. The importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics. The long term goal of our work is to provide a comprehensive understanding of the mechanism of action of this ubiquitous family of enzymes at many different length and time scales.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM051350-21A1
Application #
9235823
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter
Project Start
1994-08-01
Project End
2020-11-30
Budget Start
2017-01-01
Budget End
2017-11-30
Support Year
21
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
160079455
City
Evanston
State
IL
Country
United States
Zip Code
60201
Gunn, Kathryn H; Marko, John F; Mondragón, Alfonso (2017) An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases. Nat Struct Mol Biol 24:484-490
Brahmachari, Sumitabha; Gunn, Kathryn H; Giuntoli, Rebecca D et al. (2017) Nucleation of Multiple Buckled Structures in Intertwined DNA Double Helices. Phys Rev Lett 119:188103
Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso (2016) Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site. Nucleic Acids Res 44:3464-74
Zhang, Yan; Rajan, Rakhi; Seifert, H Steven et al. (2015) DNase H Activity of Neisseria meningitidis Cas9. Mol Cell 60:242-55
Rajan, Rakhi; Osterman, Amy K; Gast, Alexandra T et al. (2014) Biochemical characterization of the topoisomerase domain of Methanopyrus kandleri topoisomerase V. J Biol Chem 289:28898-909
Terekhova, Ksenia; Marko, John F; Mondragón, Alfonso (2014) Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III. Nucleic Acids Res 42:11657-67
Rajan, Rakhi; Prasad, Rajendra; Taneja, Bhupesh et al. (2013) Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies. Nucleic Acids Res 41:657-66
Terekhova, Ksenia; Marko, John F; Mondragon, Alfonso (2013) Studies of bacterial topoisomerases I and III at the single-molecule level. Biochem Soc Trans 41:571-5
Terekhova, Ksenia; Gunn, Kathryn H; Marko, John F et al. (2012) Bacterial topoisomerase I and topoisomerase III relax supercoiled DNA via distinct pathways. Nucleic Acids Res 40:10432-40
Baker, Nicole M; Weigand, Steven; Maar-Mathias, Sarah et al. (2011) Solution structures of DNA-bound gyrase. Nucleic Acids Res 39:755-66

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