@article{ba3d15d32cc447308bd5bb3b5ed4c29f,
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.",
keywords = "DNA replication, X-ray crystallography, native mass spectrometry, small-angle X-ray scattering",
author = "Wallen, {Jamie R.} and Hao Zhang and Caroline Weis and Weidong Cui and Foster, {Brittni M.} and Ho, {Chris M.W.} and Michal Hammel and Tainer, {John A.} and Gross, {Michael L.} and Tom Ellenberger",
note = "Funding Information: This work was supported by grants from the NIH (GM055390 to T.E., GM105404 to M.H. and J.A.T., GM103422 to M.L.G., and CA092584 to J.A.T. and T.E.) and the U.S. Department of Energy (DE-SC0001035 to M.L.G). Native MS studies were conducted at the Mass Spectrometry Facility with partial support of the Photosynthetic Antenna Research Center, an Energy Frontier Research Center funded by the U.S. Department of Energy. J.A.T. acknowledges added support of a Robert A. Welch Chemistry Chair, plus startup funds from the Cancer Prevention and Research Institute of Texas, and the University of Texas STARs program. Efforts to combine SAXS and crystallography at the SIBYLS beamline of the Advanced Light Source (Lawrence Berkeley National Laboratory) were supported in part by United States Department of Energy program IDAT. We thank our many colleagues for creative ideas and advice. Publisher Copyright: {\textcopyright} 2016 Elsevier Ltd",
year = "2017",
month = jan,
day = "3",
doi = "10.1016/j.str.2016.11.019",
language = "English",
volume = "25",
pages = "157--166",
journal = "Structure",
issn = "0969-2126",
number = "1",
}