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. 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.
authors.affiliation: Department of Chemistry & Physics, Western Carolina University, Cullowhee, NC 28723, USA. Electronic address: jamiewallen@email.wcu.edu.
authors.forename: Jamie R
authors.initials: JR
authors.lastname: Wallen
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Caroline
authors.initials: C
authors.lastname: Weis
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Weidong
authors.initials: W
authors.lastname: Cui
authors.affiliation: Department of Chemistry & Physics, Western Carolina University, Cullowhee, NC 28723, USA.
authors.forename: Brittni M
authors.initials: BM
authors.lastname: Foster
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Chris M W
authors.initials: CMW
authors.lastname: Ho
authors.affiliation: Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Michal
authors.initials: M
authors.lastname: Hammel
authors.affiliation: 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.
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: tome@biochem.wustl.edu.
authors.forename: Tom
authors.initials: T
authors.lastname: Ellenberger
mesh_terms: D017123:Bacteriophage T7 / Q000737:chemistry / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D004279:DNA, Viral; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase
keywords: DNA replication; X-ray crystallography; native mass spectrometry; small-angle X-ray scattering