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Title: Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

Abstract

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerase binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.

Authors:
 [1];  [2];  [3];  [2];  [1];  [4];  [3];  [5];  [2];  [4]
  1. Western Carolina Univ., Cullowhee, NC (United States). Dept. of Chemsitry and Physics
  2. Washington Univ., St. Louis, MO (United States). Dept. of Chemistry
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS). Molecular Biophysics and Integrated Bioimaging
  4. Washington Univ. School of Medicine, St. Louis, MO (United States). Dept. of Biochemistry and Molecular Biophysics
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS). Molecular Biophysics and Integrated Bioimaging; MD Anderson Cancer Center, Houston, TX (United States). Dept. of Molecular and Cellular Oncology
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI Identifier:
1415494
Alternate Identifier(s):
OSTI ID: 1397618; OSTI ID: 1415964
Grant/Contract Number:
AC02-05CH11231; SC0001035
Resource Type:
Journal Article: Published Article
Journal Name:
Structure
Additional Journal Information:
Journal Volume: 25; Journal Issue: 1; Journal ID: ISSN 0969-2126
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DNA replication; x-ray crystallography; native mass spectrometry; small-angle x-ray scattering

Citation Formats

Wallen, Jamie R., Zhang, Hao, Weis, Caroline, Cui, Weidong, Foster, Brittni M., Ho, Chris M. W., Hammel, Michal, Tainer, John A., Gross, Michael L., and Ellenberger, Tom. Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome. United States: N. p., 2017. Web. doi:10.1016/j.str.2016.11.019.
Wallen, Jamie R., Zhang, Hao, Weis, Caroline, Cui, Weidong, Foster, Brittni M., Ho, Chris M. W., Hammel, Michal, Tainer, John A., Gross, Michael L., & Ellenberger, Tom. Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome. United States. doi:10.1016/j.str.2016.11.019.
Wallen, Jamie R., Zhang, Hao, Weis, Caroline, Cui, Weidong, Foster, Brittni M., Ho, Chris M. W., Hammel, Michal, Tainer, John A., Gross, Michael L., and Ellenberger, Tom. Tue . "Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome". United States. doi:10.1016/j.str.2016.11.019.
@article{osti_1415494,
title = {Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome},
author = {Wallen, Jamie R. and Zhang, Hao and Weis, Caroline and Cui, Weidong and Foster, Brittni M. and Ho, Chris M. W. and Hammel, Michal and Tainer, John A. and Gross, Michael L. and Ellenberger, Tom},
abstractNote = {The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerase binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.},
doi = {10.1016/j.str.2016.11.019},
journal = {Structure},
number = 1,
volume = 25,
place = {United States},
year = {Tue Jan 03 00:00:00 EST 2017},
month = {Tue Jan 03 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.str.2016.11.019

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Cited by: 3works
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