The control of DNA topology has a major impact on the flow of genetic information. The present application focuses on type II topoisomerases, molecular machines that modulate DNA supercoiling and remove chromosome entanglements by catalyzing the ATP-dependent transport of one DNA duplex through another. Type II topoisomerases are critical for maintaining gene expression, chromosome superstructure, and genome integrity; they also serve as frontline drug targets for treating infectious disease and cancer. Progress during the prior project period, along with new data, has revealed an unanticipated richness to topoisomerase diversity and action, setting the stage for an integrated set of three aims that tackle previously unapproachable issues of how topoisomerases operate and how they can be controlled by therapeutics and biological effectors.
Aim 1 will address how ATP drives the chemomechanical action and kinetic timing of type II topoisomerases, an issue central to the function of molecular machines as a whole.
Aim 2 will establish how quinolones, one of the most successful antibiotic classes, discriminate between bacterial and human type II topoisomerases; the resulting molecular understanding of drug-protein interactions will form a framework for improving therapeutic selectivity.
Aim 3 will break new ground in the emerging and poorly- understood area of topoisomerase regulation, mechanistically defining how an prototypical representative of three different classes of effectors - natural small molecules, post-translationa modifications, and protein-protein interactions - activate or attenuate topoisomerase function. Our methodological approach is distinguished by a comprehensive blend of biochemical, structural, genetic, and single-molecule methodologies in which developments for one aim promote work on the others. Past progress and unpublished findings establish feasibility for the proposed effort. Our studies are expected to impact multiple fields, from the study of molecular machines and the control of DNA dynamics, to understanding how cells and anti-topoisomerase agents alter topoisomerase activity to support specific physiological needs and promote human health.

Public Health Relevance

Type II topoisomerases are molecular machines that disentangle DNA, as well as validated targets for frontline antibacterial and anticancer therapies. This proposal aims to understand the mechanism and regulation of topoisomerases, and to determine how some of the most widely-used anti-topoisomerase drugs block enzyme function.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA077373-20
Application #
9209950
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Knowlton, John R
Project Start
1999-05-01
Project End
2019-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
20
Fiscal Year
2017
Total Cost
$400,843
Indirect Cost
$127,854
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205
Hauk, Glenn; Berger, James M (2016) The role of ATP-dependent machines in regulating genome topology. Curr Opin Struct Biol 36:85-96
Aldred, Katie J; Blower, Tim R; Kerns, Robert J et al. (2016) Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase. Proc Natl Acad Sci U S A 113:E839-46
Blower, Tim R; Williamson, Benjamin H; Kerns, Robert J et al. (2016) Crystal structure and stability of gyrase-fluoroquinolone cleaved complexes from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 113:1706-13
Vos, Seychelle M; Lyubimov, Artem Y; Hershey, David M et al. (2014) Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein. Genes Dev 28:1485-97
Drlica, Karl; Mustaev, Arkady; Towle, Tyrell R et al. (2014) Bypassing fluoroquinolone resistance with quinazolinediones: studies of drug-gyrase-DNA complexes having implications for drug design. ACS Chem Biol 9:2895-904
Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin et al. (2014) Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding. J Biol Chem 289:12300-12
Kranzusch, Philip J; Lee, Amy S Y; Wilson, Stephen C et al. (2014) Structure-guided reprogramming of human cGAS dinucleotide linkage specificity. Cell 158:1011-21
Vos, Seychelle M; Lee, Imsang; Berger, James M (2013) Distinct regions of the Escherichia coli ParC C-terminal domain are required for substrate discrimination by topoisomerase IV. J Mol Biol 425:3029-45
Vos, Seychelle M; Stewart, Nichole K; Oakley, Martha G et al. (2013) Structural basis for the MukB-topoisomerase IV interaction and its functional implications in vivo. EMBO J 32:2950-62
Tretter, Elsa M; Berger, James M (2012) Mechanisms for defining supercoiling set point of DNA gyrase orthologs: II. The shape of the GyrA subunit C-terminal domain (CTD) is not a sole determinant for controlling supercoiling efficiency. J Biol Chem 287:18645-54

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