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Structure. 2017 Jan 3;25(1):157-166. doi: 10.1016/j.str.2016.11.019.

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

Author information

1
Department of Chemistry & Physics, Western Carolina University, Cullowhee, NC 28723, USA. Electronic address: jamiewallen@email.wcu.edu.
2
Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA.
3
Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
4
Department of Chemistry & Physics, Western Carolina University, Cullowhee, NC 28723, USA.
5
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
6
Department of Molecular and Cellular Oncology, MD Anderson Cancer Center, Houston, TX 77054, USA; Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
7
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: tome@biochem.wustl.edu.

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. 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. Our collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.

KEYWORDS:

DNA replication; X-ray crystallography; native mass spectrometry; small-angle X-ray scattering

PMID:
28052235
PMCID:
PMC5267931
DOI:
10.1016/j.str.2016.11.019
[Indexed for MEDLINE]
Free PMC Article

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