Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.pdf (2.26 MB)
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Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.

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posted on 27.11.2018, 11:30 by Jiangyu Yan, Thomas R. Beattie, Adriana L. Rojas, Kelly Schermerhorn, Tamzin Gristwood, Jonathan C. Trinidad, Sonja V. Albers, Pietro Roversi, Andrew F. Gardner, Nicola G. A. Abrescia, Stephen D. Bell
Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.

Funding

This investigation has been supported by funds from the College of Arts and Sciences, Indiana University to S.D.B and by MINECO/FEDER (BFU2015-64541-R) to N.G.A.A. P.R. was a recipient of the Basque Foundation for Science Visiting Fellowship 2012. J.Y is supported by the College of Arts and Sciences, Indiana University. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X grant agreements n° 283570.

History

Citation

Nature Communications, 2017, 8, 15075

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Molecular & Cell Biology

Version

VoR (Version of Record)

Published in

Nature Communications

Publisher

Nature Research (part of Springer Nature)

eissn

2041-1723

Acceptance date

27/02/2017

Copyright date

2017

Available date

27/11/2018

Publisher version

https://www.nature.com/articles/ncomms15075

Notes

Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Protein Data Bank: Atomic coordinates and structure factors have been deposited under the accession number 5N35 (Gd-PBP2) and 5N41 (PBP2). Electron Microscopy Bank: Maps corresponding to the Apo and holo structures have been deposited with the accession numbers EMD-3458 (apo-PolB1), EMD3462 (PolB1-HE).

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en

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