skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases

Abstract

Abstract OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC−Escherichia coli hosts, indicating that both the ATPase and nucleasemore » activities are required for OLD function in vivo.« less

Authors:
 [1]; ORCiD logo [1]; ORCiD logo [1]
  1. Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1596875
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Published Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Name: Nucleic Acids Research; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Schiltz, Carl J., Adams, Myfanwy C., and Chappie, Joshua S. The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases. United Kingdom: N. p., 2020. Web. doi:10.1093/nar/gkaa059.
Schiltz, Carl J., Adams, Myfanwy C., & Chappie, Joshua S. The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases. United Kingdom. doi:10.1093/nar/gkaa059.
Schiltz, Carl J., Adams, Myfanwy C., and Chappie, Joshua S. Mon . "The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases". United Kingdom. doi:10.1093/nar/gkaa059.
@article{osti_1596875,
title = {The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases},
author = {Schiltz, Carl J. and Adams, Myfanwy C. and Chappie, Joshua S.},
abstractNote = {Abstract OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC−Escherichia coli hosts, indicating that both the ATPase and nuclease activities are required for OLD function in vivo.},
doi = {10.1093/nar/gkaa059},
journal = {Nucleic Acids Research},
number = ,
volume = ,
place = {United Kingdom},
year = {2020},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1093/nar/gkaa059

Save / Share:

Works referenced in this record:

Role of the D-Loops in Allosteric Control of ATP Hydrolysis in an ABC Transporter
journal, December 2011

  • Jones, Peter M.; George, Anthony M.
  • The Journal of Physical Chemistry A, Vol. 116, Issue 11
  • DOI: 10.1021/jp211139s

The crystal structure of DNA mismatch repair protein MutS binding to a G·T mismatch
journal, October 2000

  • Lamers, Meindert H.; Perrakis, Anastassis; Enzlin, Jacqueline H.
  • Nature, Vol. 407, Issue 6805
  • DOI: 10.1038/35037523

RecA-like motor ATPases—lessons from structures
journal, November 2004

  • Ye, Jiqing; Osborne, Andrew R.; Groll, Michael
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1659, Issue 1
  • DOI: 10.1016/j.bbabio.2004.06.003

ATP-dependent conformational change in ABC-ATPase RecF serves as a switch in DNA repair
journal, February 2018


Bacteriophage Lambda; Abortive Infection of Bacteria Lysogenic for Phage P2
journal, July 1970

  • Lindahl, G.; Sironi, G.; Bialy, H.
  • Proceedings of the National Academy of Sciences, Vol. 66, Issue 3
  • DOI: 10.1073/pnas.66.3.587

Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium
journal, February 2012

  • Khatiwara, Anita; Jiang, Tieshan; Sung, Sam-Sun
  • Applied and Environmental Microbiology, Vol. 78, Issue 9
  • DOI: 10.1128/AEM.06865-11

Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA
journal, October 2000

  • Obmolova, Galina; Ban, Changill; Hsieh, Peggy
  • Nature, Vol. 407, Issue 6805
  • DOI: 10.1038/35037509

A short history of SHELX
journal, December 2007

  • Sheldrick, George M.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 64, Issue 1, p. 112-122
  • DOI: 10.1107/S0108767307043930

Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage
journal, August 2019

  • Schiltz, Carl J.; Lee, April; Partlow, Edward A.
  • Nucleic Acids Research, Vol. 47, Issue 17
  • DOI: 10.1093/nar/gkz703

Dali server: conservation mapping in 3D
journal, May 2010

  • Holm, Liisa; Rosenstr�m, P�ivi
  • Nucleic Acids Research, Vol. 38, Issue suppl_2
  • DOI: 10.1093/nar/gkq366

Distinct Structural and Functional Properties of the ATPase Sites in an Asymmetric ABC Transporter
journal, October 2006


Anomalous diffraction in crystallographic phase evaluation
journal, February 2014


The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle
journal, October 2014

  • Zolnerciks, Joseph K.; Akkaya, Begum G.; Snippe, Marjolein
  • The FASEB Journal, Vol. 28, Issue 10
  • DOI: 10.1096/fj.13-245639

Structure and mechanism of ABC transporter proteins
journal, August 2007

  • Hollenstein, Kaspar; Dawson, Roger JP; Locher, Kaspar P.
  • Current Opinion in Structural Biology, Vol. 17, Issue 4
  • DOI: 10.1016/j.sbi.2007.07.003

In Vivo Studies of Temperature-Sensitive recB and recC Mutants
journal, January 1974


H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB
journal, May 2005

  • Zaitseva, Jelena; Jenewein, Stefan; Jumpertz, Thorsten
  • The EMBO Journal, Vol. 24, Issue 11
  • DOI: 10.1038/sj.emboj.7600657

Nucleases: diversity of structure, function and mechanism
journal, September 2010


Structure of Full-Length SMC and Rearrangements Required for Chromosome Organization
journal, July 2017


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

ATP ‐dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex
journal, December 2015


Mechanism of formation of a toroid around DNA by the mismatch sensor protein
journal, November 2017

  • Nirwal, Shivlee; Kulkarni, Dhananjaya S.; Sharma, Amit
  • Nucleic Acids Research, Vol. 46, Issue 1
  • DOI: 10.1093/nar/gkx1149

Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
journal, March 2017

  • Deshpande, Rajashree A.; Lee, Ji-Hoon; Paull, Tanya T.
  • Nucleic Acids Research, Vol. 45, Issue 9
  • DOI: 10.1093/nar/gkx173

Invited review: Architectures and mechanisms of ATP binding cassette proteins
journal, May 2016


Snapshots of the maltose transporter during ATP hydrolysis
journal, August 2011

  • Oldham, M. L.; Chen, J.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 37
  • DOI: 10.1073/pnas.1108858108

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50
journal, May 2011

  • De la Rosa, Metzere Bierlein; Nelson, Scott W.
  • Journal of Biological Chemistry, Vol. 286, Issue 29
  • DOI: 10.1074/jbc.M111.256305

Structure of a Pancreatic ATP-Sensitive Potassium Channel
journal, January 2017


A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair
journal, January 2018


The Rad50 Signature Motif: Essential to ATP Binding and Biological Function
journal, January 2004

  • Moncalian, Gabriel; Lengsfeld, Bettina; Bhaskara, Venugopal
  • Journal of Molecular Biology, Vol. 335, Issue 4
  • DOI: 10.1016/j.jmb.2003.11.026

Scaling and assessment of data quality
journal, December 2005

  • Evans, Philip
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 1, p. 72-82
  • DOI: 10.1107/S0907444905036693

ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules
journal, May 2016

  • Ashkenazy, Haim; Abadi, Shiran; Martz, Eric
  • Nucleic Acids Research, Vol. 44, Issue W1
  • DOI: 10.1093/nar/gkw408

Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli
journal, August 2016

  • Stracy, Mathew; Jaciuk, Marcin; Uphoff, Stephan
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms12568

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

Structure of the Human MutSα DNA Lesion Recognition Complex
journal, May 2007


Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function
journal, January 2007

  • Koroleva, Olga; Makharashvili, Nodar; Courcelle, Charmain T.
  • The EMBO Journal, Vol. 26, Issue 3
  • DOI: 10.1038/sj.emboj.7601537

CFTR structure and cystic fibrosis
journal, July 2014

  • Cant, Natasha; Pollock, Naomi; Ford, Robert C.
  • The International Journal of Biochemistry & Cell Biology, Vol. 52
  • DOI: 10.1016/j.biocel.2014.02.004