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 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 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 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 scales, from the atomic level to the nano scale. In the past period we made substantial progress towards this goal, including solving atomic structures of topoisomerase III in complex with DNA, a structure of D. radiodurans topoisomerase IB in complex with DNA that revealed the existence of a secondary site, structures of several fragments of topoisomerase V, and single molecule studies of topoisomerase V. In addition, we continue making progress with our single molecule studies of topoisomerases and also initiated work on a complex of gyrase with a large DNA fragment. Our studies are providing important information on these molecules and allowing us to relate the 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 study the mechanism of type IA topoisomerases at the single molecule level, ii) to study the structure and catalytic mechanism of topoisomerase V, the topoisomerase most recently discovered, and iii) to study the structure of a complex of gyrase with DNA. 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.
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 scales, from the atomic level to the nano scale.
|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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