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 DMA by transiently breaking one (type I) or two (type II) DMA strands and passing another single or double strand through the break. The study of the structure and function of topoisomerases promises not only to further our understanding of proteins that interact with DMA and alter its topological properties, but also to provide important information to aid in the design of new therapeutic agents. Type I enzymes have been sub-classified into two different families, types IA and IB, depending on whether they form a transient covalent bond with the 5'or 3'end of the broken DMA strand. Recently, we solved the structure of archaeal topoisomerase V and discovered that it represents a new subtype with a distinct fold and a different catalytic mechanism. This discovery has changed our understanding of all topoisomerases. Aside from the work on topoisomerase V, we also made substantial progress in our studies of other type I topoisomerases. We solved structures of two type IA enzymes in complex with DMA and solved the structure of D. radiodurans topoisomerase IB, a newly discovered bacterial type IB topoisomerase, alone and in a non-specific complex with DMA.
The specific aims of this proposal are: i) to study the structure and function of type IA topoisomerases, ii) to study the structure and function of viral and bacterial type IB topoisomerases, iii) to study the structure of complexes of topoisomerase V with DNA, iv) to characterize the catalytic mechanism of DNA cleavage/religation and DNA repair by topoisomerase V, and v) to elucidate the mechanism of DNA relaxation employed by topoisomerase V using single molecule techniques. 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 to solve their structures to high resolution, and single molecule studies to elucidate their mechanism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051350-16
Application #
7796675
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
1994-08-01
Project End
2011-04-30
Budget Start
2010-04-01
Budget End
2011-04-30
Support Year
16
Fiscal Year
2010
Total Cost
$258,554
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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