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Title: Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase

Abstract

Abstract Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

Authors:
ORCiD logo [1]; ORCiD logo [1];  [1]; ORCiD logo [2]
  1. Departments of Pharmaceutical Sciences, University of California, Irvine, CA 92697-3958, USA
  2. Departments of Pharmaceutical Sciences, University of California, Irvine, CA 92697-3958, USA, Department of Chemistry, University of California, Irvine, CA 92697-3958, USA, Department of Molecular Biology and Biochemistry, University of California, CA 92697-3958, USA
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1525514
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Published Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Name: Nucleic Acids Research Journal Volume: 47 Journal Issue: 13; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Jackson, Lynnette N., Chim, Nicholas, Shi, Changhua, and Chaput, John C. Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase. United Kingdom: N. p., 2019. Web. doi:10.1093/nar/gkz513.
Jackson, Lynnette N., Chim, Nicholas, Shi, Changhua, & Chaput, John C. Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase. United Kingdom. doi:10.1093/nar/gkz513.
Jackson, Lynnette N., Chim, Nicholas, Shi, Changhua, and Chaput, John C. Thu . "Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase". United Kingdom. doi:10.1093/nar/gkz513.
@article{osti_1525514,
title = {Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase},
author = {Jackson, Lynnette N. and Chim, Nicholas and Shi, Changhua and Chaput, John C.},
abstractNote = {Abstract Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.},
doi = {10.1093/nar/gkz513},
journal = {Nucleic Acids Research},
number = 13,
volume = 47,
place = {United Kingdom},
year = {2019},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1093/nar/gkz513

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Works referenced in this record:

Structural Insights into the Processing of Nucleobase-Modified Nucleotides by DNA Polymerases
journal, February 2016


TM-align: a protein structure alignment algorithm based on the TM-score
journal, April 2005


Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues
journal, April 2018

  • Mei, Hui; Liao, Jen-Yu; Jimenez, Randi M.
  • Journal of the American Chemical Society, Vol. 140, Issue 17
  • DOI: 10.1021/jacs.7b13031

Chemical discrimination between dC and 5Me dC via their hydroxylamine adducts
journal, September 2010

  • Münzel, Martin; Lercher, Lukas; Müller, Markus
  • Nucleic Acids Research, Vol. 38, Issue 21
  • DOI: 10.1093/nar/gkq724

Synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue
journal, May 2017

  • Mei, Hui; Shi, Changhua; Jimenez, Randi M.
  • Nucleic Acids Research, Vol. 45, Issue 10
  • DOI: 10.1093/nar/gkx368

Web 3DNA--a web server for the analysis, reconstruction, and visualization of three-dimensional nucleic-acid structures
journal, May 2009

  • Zheng, G.; Lu, X. -J.; Olson, W. K.
  • Nucleic Acids Research, Vol. 37, Issue Web Server
  • DOI: 10.1093/nar/gkp358

Structures of KlenTaq DNA Polymerase Caught While Incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates
journal, April 2012

  • Bergen, Konrad; Steck, Anna-Lena; Strütt, Stefan
  • Journal of the American Chemical Society, Vol. 134, Issue 29
  • DOI: 10.1021/ja3017889

Synthetic Genetic Polymers Capable of Heredity and Evolution
journal, April 2012


Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP
journal, June 1994


DNA Polymerase-Mediated DNA Synthesis on a TNA Template
journal, January 2003

  • Chaput, John C.; Ichida, Justin K.; Szostak, Jack W.
  • Journal of the American Chemical Society, Vol. 125, Issue 4
  • DOI: 10.1021/ja028589k

Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution
journal, January 1998

  • Doublié, Sylvie; Tabor, Stanley; Long, Alexander M.
  • Nature, Vol. 391, Issue 6664
  • DOI: 10.1038/34593

Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity.
journal, December 1997


Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase
journal, August 2016


Replicative DNA Polymerases
journal, June 2013


Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations
journal, March 2003

  • Johnson, S. J.; Taylor, J. S.; Beese, L. S.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 7
  • DOI: 10.1073/pnas.0630532100

Active Site Tightness and Substrate Fit in DNA Replication
journal, June 2002


Towards automated crystallographic structure refinement with phenix.refine
journal, March 2012

  • Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
  • DOI: 10.1107/S0907444912001308

Reverse transcription and direct amplification of cellular RNA transcripts by Taq polymerase
journal, April 1990


The solution structure of double helical arabino nucleic acids (ANA and 2′F-ANA): effect of arabinoses in duplex-hairpin interconversion
journal, July 2012

  • Martín-Pintado, Nerea; Yahyaee-Anzahaee, Maryam; Campos-Olivas, Ramón
  • Nucleic Acids Research, Vol. 40, Issue 18
  • DOI: 10.1093/nar/gks672

Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution
journal, January 1997


XDS
journal, January 2010

  • Kabsch, Wolfgang
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2
  • DOI: 10.1107/S0907444909047337

A unified polymerase mechanism for nonhomologous DNA and RNA polymerases
journal, December 1994


UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes
journal, July 2016

  • Anosova, Irina; Kowal, Ewa A.; Sisco, Nicholas J.
  • ChemBioChem, Vol. 17, Issue 18
  • DOI: 10.1002/cbic.201600349

The structural diversity of artificial genetic polymers
journal, December 2015

  • Anosova, Irina; Kowal, Ewa A.; Dunn, Matthew R.
  • Nucleic Acids Research, Vol. 44, Issue 3
  • DOI: 10.1093/nar/gkv1472

Why Does TNA Cross-Pair More Strongly with RNA Than with DNA? An Answer From X-ray Analysis
journal, December 2003

  • Pallan, Pradeep S.; Wilds, Christopher J.; Wawrzak, Zdzislaw
  • Angewandte Chemie International Edition, Vol. 42, Issue 47
  • DOI: 10.1002/anie.200352553

A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity
journal, January 1991

  • Carroll, Steven S.; Cowart, Marlon; Benkovic, Stephen J.
  • Biochemistry, Vol. 30, Issue 3
  • DOI: 10.1021/bi00217a034

Snapshots of a modified nucleotide moving through the confines of a DNA polymerase
journal, September 2018

  • Kropp, Heike Maria; Dürr, Simon Leonard; Peter, Christine
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 40
  • DOI: 10.1073/pnas.1811518115

The Structure of a TNA−TNA Complex in Solution: NMR Study of the Octamer Duplex Derived from α-( l )-Threofuranosyl-(3′-2′)-CGAATTCG
journal, November 2008

  • Ebert, Marc-Olivier; Mang, Christian; Krishnamurthy, Ramanarayanan
  • Journal of the American Chemical Society, Vol. 130, Issue 45
  • DOI: 10.1021/ja8041959

Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers
journal, April 2012


iMOSFLM : a new graphical interface for diffraction-image processing with MOSFLM
journal, March 2011

  • Battye, T. Geoff G.; Kontogiannis, Luke; Johnson, Owen
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444910048675

Capture of a third Mg2+ is essential for catalyzing DNA synthesis
journal, June 2016


Structures of Mismatch Replication Errors Observed in a DNA Polymerase
journal, March 2004


Analysis of aptamer discovery and technology
journal, October 2017

  • Dunn, Matthew R.; Jimenez, Randi M.; Chaput, John C.
  • Nature Reviews Chemistry, Vol. 1, Issue 10
  • DOI: 10.1038/s41570-017-0076

Structures of KOD and 9°N DNA Polymerases Complexed with Primer Template Duplex
journal, June 2013


Evolution of a General RNA-Cleaving FANA Enzyme
journal, November 2018


“Skinny” and “Fat” DNA: Two New Double Helices
journal, August 2018

  • Hoshika, Shuichi; Singh, Isha; Switzer, Christopher
  • Journal of the American Chemical Society, Vol. 140, Issue 37
  • DOI: 10.1021/jacs.8b05042

Replication of hexitol oligonucleotides as a prelude to the propagation of a third type of nucleic acid in vivo
journal, December 2003

  • Pochet, Sylvie; Kaminski, P. Alexandre; Van Aerschot, Arthur
  • Comptes Rendus Biologies, Vol. 326, Issue 12
  • DOI: 10.1016/j.crvi.2003.10.004

MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009

  • Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
  • DOI: 10.1107/S0907444909042073

Base Miscoding and Strand Misalignment Errors by Mutator Klenow Polymerases with Amino Acid Substitutions at Tyrosine 766 in the O Helix of the Fingers Subdomain
journal, March 1997

  • Bell, Juliette B.; Eckert, Kristin A.; Joyce, Catherine M.
  • Journal of Biological Chemistry, Vol. 272, Issue 11
  • DOI: 10.1074/jbc.272.11.7345

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

Crystal Structure of a B-Form DNA Duplex Containing ( l )-α-Threofuranosyl (3‘→2‘) Nucleosides:  A Four-Carbon Sugar Is Easily Accommodated into the Backbone of DNA
journal, November 2002

  • Wilds, Christopher J.; Wawrzak, Zdzislaw; Krishnamurthy, Ramanarayanan
  • Journal of the American Chemical Society, Vol. 124, Issue 46
  • DOI: 10.1021/ja0207807

DNA polymerases and biotechnological applications
journal, December 2017


Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal
journal, January 1998

  • Kiefer, James R.; Mao, Chen; Braman, Jeffrey C.
  • Nature, Vol. 391, Issue 6664
  • DOI: 10.1038/34693

Engineering and application of polymerases for synthetic genetics
journal, December 2017

  • Houlihan, Gillian; Arangundy-Franklin, Sebastian; Holliger, Philipp
  • Current Opinion in Biotechnology, Vol. 48
  • DOI: 10.1016/j.copbio.2017.04.004

2‘,5‘-Linked DNA Is a Template for Polymerase-Directed DNA Synthesis
journal, January 2004

  • Sinha, Surajit; Kim, Paul H.; Switzer, Christopher
  • Journal of the American Chemical Society, Vol. 126, Issue 1
  • DOI: 10.1021/ja034986z

Innate Reverse Transcriptase Activity of DNA Polymerase for Isothermal RNA Direct Detection
journal, October 2015

  • Shi, Chao; Shen, Xiaotong; Niu, Shuyan
  • Journal of the American Chemical Society, Vol. 137, Issue 43
  • DOI: 10.1021/jacs.5b08144

A Scalable Synthesis of α- l -Threose Nucleic Acid Monomers
journal, February 2016

  • Sau, Sujay P.; Fahmi, Nour Eddine; Liao, Jen-Yu.
  • The Journal of Organic Chemistry, Vol. 81, Issue 6
  • DOI: 10.1021/acs.joc.5b02768

Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway
journal, October 2018


Thermostable Bst DNA polymerase I lacks a 3′ → 5′ proofreading exonuclease activity
journal, March 1996

  • Aliotta, Jason M.; Pelletier, John J.; Ware, Jennifer L.
  • Genetic Analysis: Biomolecular Engineering, Vol. 12, Issue 5-6
  • DOI: 10.1016/S1050-3862(96)80005-2

Unlocking the Sugar “Steric Gate” of DNA Polymerases
journal, February 2011

  • Brown, Jessica A.; Suo, Zucai
  • Biochemistry, Vol. 50, Issue 7
  • DOI: 10.1021/bi101915z

Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between product and template
journal, September 2007

  • Tsai, C. -H.; Chen, J.; Szostak, J. W.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 37
  • DOI: 10.1073/pnas.0704211104

A general strategy for expanding polymerase function by droplet microfluidics
journal, April 2016

  • Larsen, Andrew C.; Dunn, Matthew R.; Hatch, Andrew
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms11235

Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids
journal, November 2012


The Emerging World of Synthetic Genetics
journal, November 2012


Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206

Evaluating TNA stability under simulated physiological conditions
journal, May 2016

  • Culbertson, Michelle C.; Temburnikar, Kartik W.; Sau, Sujay P.
  • Bioorganic & Medicinal Chemistry Letters, Vol. 26, Issue 10
  • DOI: 10.1016/j.bmcl.2016.03.118