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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter C
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Northwestern University at Chicago
Schools of Arts and Sciences
United States
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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
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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
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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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