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Title: Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site

Abstract

Here, we have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drugmore » target.« less

Authors:
 [1];  [2];  [1];  [2];  [1]
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  1. Florida Intl Univ., Miami, FL (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
National Institutes of Health (NIH); Bill and Melinda Gates Foundation; USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1491852
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 46; Journal Issue: 14; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Cao, Nan, Tan, Kemin, Annamalai, Thirunavukkarasu, Joachimiak, Andrzej, and Tse-Dinh, Yuk -Ching. Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site. United States: N. p., 2018. Web. doi:10.1093/nar/gky492.
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Cao, Nan, Tan, Kemin, Annamalai, Thirunavukkarasu, Joachimiak, Andrzej, & Tse-Dinh, Yuk -Ching. Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site. United States. https://doi.org/10.1093/nar/gky492
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Cao, Nan, Tan, Kemin, Annamalai, Thirunavukkarasu, Joachimiak, Andrzej, and Tse-Dinh, Yuk -Ching. Thu . "Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site". United States. https://doi.org/10.1093/nar/gky492. https://www.osti.gov/servlets/purl/1491852.
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@article{osti_1491852,
title = {Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site},
author = {Cao, Nan and Tan, Kemin and Annamalai, Thirunavukkarasu and Joachimiak, Andrzej and Tse-Dinh, Yuk -Ching},
abstractNote = {Here, we have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.},
doi = {10.1093/nar/gky492},
journal = {Nucleic Acids Research},
number = 14,
volume = 46,
place = {United States},
year = {Thu Jun 14 00:00:00 EDT 2018},
month = {Thu Jun 14 00:00:00 EDT 2018}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1093/nar/gky492
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Cited by: 15 works
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Table 1 Table 1: Data collection and refinement statistics
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    Works referencing / citing this record:

    Direct observation of topoisomerase IA gate dynamics
    journal, November 2018

    • Mills, Maria; Tse-Dinh, Yuk-Ching; Neuman, Keir C.
    • Nature Structural & Molecular Biology, Vol. 25, Issue 12
    • DOI: 10.1038/s41594-018-0158-x

    Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound
    journal, February 2019

    Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound
    text, January 2019

    • Garcia, Pamela K.; Annamalai, Thirunavukkarasu; Wang, Wenjie
    • Columbia University
    • DOI: 10.7916/d8-r8fb-gy22
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