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Title: The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

Abstract

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2more » initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.« less

Authors:
 [1];  [2];  [3];  [1];  [4];  [1]
+ Show Author Affiliations
  1. National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst. (NCI), Center for Cancer Research (CCR)
  2. National Cancer Inst., Frederick, MD (United States). Macromolecular Crystallography Lab.; Frederick National Lab. for Cancer Research, MD (United States). Leidos Biomedical Research, Basic Science Program
  3. National Inst. of Health (NIH), Bethesda, MD (United States). National Inst. of Diabetes, Digestive and Kidney Diseases (NIDDK)
  4. National Cancer Inst., Frederick, MD (United States). Macromolecular Crystallography Lab.
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); National Cancer Institute (NCI); Center for Cancer Research; National Institute of Diabetes, Digestive and Kidney Diseases
OSTI Identifier:
1355049
Grant/Contract Number:  
W-31-109-Eng-38
Resource Type:
Accepted Manuscript
Journal Name:
mBio (Online)
Additional Journal Information:
Journal Name: mBio (Online); Journal Volume: 8; Journal Issue: 2; Journal ID: ISSN 2150-7511
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; chromosome replication; DNA-protein interactions; DnaK chaperone; initiator remodeling; initiator structure; Vibrio cholerae

Citation Formats

Jha, Jyoti K., Li, Mi, Ghirlando, Rodolfo, Miller Jenkins, Lisa M., Wlodawer, Alexander, and Chattoraj, Dhruba. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2. United States: N. p., 2017. Web. doi:10.1128/mBio.00427-17.
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Jha, Jyoti K., Li, Mi, Ghirlando, Rodolfo, Miller Jenkins, Lisa M., Wlodawer, Alexander, & Chattoraj, Dhruba. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2. United States. https://doi.org/10.1128/mBio.00427-17
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Jha, Jyoti K., Li, Mi, Ghirlando, Rodolfo, Miller Jenkins, Lisa M., Wlodawer, Alexander, and Chattoraj, Dhruba. Tue . "The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2". United States. https://doi.org/10.1128/mBio.00427-17. https://www.osti.gov/servlets/purl/1355049.
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@article{osti_1355049,
title = {The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2},
author = {Jha, Jyoti K. and Li, Mi and Ghirlando, Rodolfo and Miller Jenkins, Lisa M. and Wlodawer, Alexander and Chattoraj, Dhruba},
abstractNote = {Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.},
doi = {10.1128/mBio.00427-17},
journal = {mBio (Online)},
number = 2,
volume = 8,
place = {United States},
year = {Tue Apr 18 00:00:00 EDT 2017},
month = {Tue Apr 18 00:00:00 EDT 2017}
}
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    Works referencing / citing this record:

    Vibrio cholerae chromosome 2 copy number is controlled by the methylation-independent binding of its monomeric initiator to the chromosome 1 crtS site
    journal, September 2018

    • de Lemos Martins, Francisco; Fournes, Florian; Mazzuoli, Maria-Vittoria
    • Nucleic Acids Research
    • DOI: 10.1093/nar/gky790
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