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

Title: Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS

Abstract

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior gives a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]
  1. National Inst. of Health (NIH), Research Triangle Park, NC (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1213717
Grant/Contract Number:  
AC02-06CH11357; W-31-109-ENG-38
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 5; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; protein translocation; x-ray crystallography; DNA damage and repair

Citation Formats

Kirby, Thomas W., Gassman, Natalie R., Smith, Cassandra E., Pedersen, Lars C., Gabel, Scott A., Sobhany, Mack, Wilson, Samuel H., and London, Robert E. Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS. United States: N. p., 2015. Web. doi:10.1038/srep13405.
Kirby, Thomas W., Gassman, Natalie R., Smith, Cassandra E., Pedersen, Lars C., Gabel, Scott A., Sobhany, Mack, Wilson, Samuel H., & London, Robert E. Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS. United States. doi:10.1038/srep13405.
Kirby, Thomas W., Gassman, Natalie R., Smith, Cassandra E., Pedersen, Lars C., Gabel, Scott A., Sobhany, Mack, Wilson, Samuel H., and London, Robert E. Tue . "Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS". United States. doi:10.1038/srep13405. https://www.osti.gov/servlets/purl/1213717.
@article{osti_1213717,
title = {Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS},
author = {Kirby, Thomas W. and Gassman, Natalie R. and Smith, Cassandra E. and Pedersen, Lars C. and Gabel, Scott A. and Sobhany, Mack and Wilson, Samuel H. and London, Robert E.},
abstractNote = {We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior gives a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.},
doi = {10.1038/srep13405},
journal = {Scientific Reports},
issn = {2045-2322},
number = ,
volume = 5,
place = {United States},
year = {2015},
month = {8}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 10 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Single-strand break repair and genetic disease
journal, August 2008

  • Caldecott, Keith W.
  • Nature Reviews Genetics, Vol. 9, Issue 8
  • DOI: 10.1038/nrg2380

DNA single-strand break repair
journal, November 2014


Sealing of Chromosomal DNA Nicks during Nucleotide Excision Repair Requires XRCC1 and DNA Ligase IIIα in a Cell-Cycle-Specific Manner
journal, July 2007


Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells
journal, March 2010


XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage
journal, June 1998

  • Masson, Murielle; Niedergang, Claude; Schreiber, Valérie
  • Molecular and Cellular Biology, Vol. 18, Issue 6
  • DOI: 10.1128/MCB.18.6.3563

Central Role for the XRCC1 BRCT I Domain in Mammalian DNA Single-Strand Break Repair
journal, April 2002


DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair
journal, December 2014

  • Abdou, Ismail; Poirier, Guy G.; Hendzel, Michael J.
  • Nucleic Acids Research, Vol. 43, Issue 2
  • DOI: 10.1093/nar/gku1307

Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair
journal, January 2013

  • Campalans, Anna; Kortulewski, Thierry; Amouroux, Rachel
  • Nucleic Acids Research, Vol. 41, Issue 5
  • DOI: 10.1093/nar/gkt025

Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate
journal, December 2010

  • Strom, C. E.; Johansson, F.; Uhlen, M.
  • Nucleic Acids Research, Vol. 39, Issue 8
  • DOI: 10.1093/nar/gkq1241

Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α
journal, December 2006

  • Lange, Allison; Mills, Ryan E.; Lange, Christopher J.
  • Journal of Biological Chemistry, Vol. 282, Issue 8
  • DOI: 10.1074/jbc.R600026200

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope
journal, April 1995


Restoration of nuclear-import failure caused by triple A syndrome and oxidative stress
journal, October 2008

  • Kiriyama, Takao; Hirano, Makito; Asai, Hirohide
  • Biochemical and Biophysical Research Communications, Vol. 374, Issue 4
  • DOI: 10.1016/J.Bbrc.2008.07.088

Novel Binding of the Mitotic Regulator TPX2 (Target Protein for Xenopus Kinesin-like Protein 2) to Importin-α
journal, March 2010

  • Giesecke, Astrid; Stewart, Murray
  • Journal of Biological Chemistry, Vol. 285, Issue 23
  • DOI: 10.1074/jbc.M110.102343

Six Classes of Nuclear Localization Signals Specific to Different Binding Grooves of Importin α
journal, November 2008

  • Kosugi, Shunichi; Hasebe, Masako; Matsumura, Nobutaka
  • Journal of Biological Chemistry, Vol. 284, Issue 1
  • DOI: 10.1074/jbc.M807017200

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal
journal, March 2010


Molecular basis for specificity of nuclear import and prediction of nuclear localization
journal, September 2011

  • Marfori, Mary; Mynott, Andrew; Ellis, Jonathan J.
  • Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1813, Issue 9
  • DOI: 10.1016/j.bbamcr.2010.10.013

An actin-regulated importin α/β-dependent extended bipartite NLS directs nuclear import of MRTF-A
journal, September 2010

  • Pawłowski, Rafał; Rajakylä, Eeva Kaisa; Vartiainen, Maria K.
  • The EMBO Journal, Vol. 29, Issue 20
  • DOI: 10.1038/emboj.2010.216

Nuclear Import of Chromatin Remodeler Isw1 Is Mediated by Atypical Bipartite cNLS and Classical Import Pathway: Nuclear Import of Isw1
journal, November 2012

  • Vasicova, Pavla; Stradalova, Vendula; Halada, Petr
  • Traffic, Vol. 14, Issue 2
  • DOI: 10.1111/tra.12025

An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity
journal, February 2003


The Human DNA Ligase III Gene Encodes Nuclear and Mitochondrial Proteins
journal, May 1999

  • Lakshmipathy, Uma; Campbell, Colin
  • Molecular and Cellular Biology, Vol. 19, Issue 5
  • DOI: 10.1128/MCB.19.5.3869

Eukaryotic DNA Ligases: Structural and Functional Insights
journal, June 2008


XRCC1 Protein Interacts with One of Two Distinct Forms of DNA Ligase III
journal, April 1997

  • Nash, Rachel A.; Caldecott, Keith W.; Barnes, Deborah E.
  • Biochemistry, Vol. 36, Issue 17
  • DOI: 10.1021/Bi962281m

JWA regulates XRCC1 and functions as a novel base excision repair protein in oxidative-stress-induced DNA single-strand breaks
journal, February 2009

  • Wang, Shouyu; Gong, Zhenghua; Chen, Rui
  • Nucleic Acids Research, Vol. 37, Issue 6
  • DOI: 10.1093/Nar/Gkp054

Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-α11Edited by K. Nagai
journal, April 2000

  • Fontes, Marcos R. M.; Teh, Trazel; Kobe, Bostjan
  • Journal of Molecular Biology, Vol. 297, Issue 5
  • DOI: 10.1006/jmbi.2000.3642

Allosteric Receptors after 30 Years
journal, November 1998


Nuclear translocation contributes to regulation of DNA excision repair activities
journal, June 2009


The structural basis of XRCC1-mediated DNA repair
journal, June 2015


Recognition and repair of chemically heterogeneous structures at DNA ends: Detection and Repair of Abnormal DNA Termini
journal, August 2014

  • Andres, Sara N.; Schellenberg, Matthew J.; Wallace, Bret D.
  • Environmental and Molecular Mutagenesis, Vol. 56, Issue 1
  • DOI: 10.1002/em.21892

XRCC1 and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions
journal, January 2007


PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization
journal, January 1999


NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction
journal, January 2009

  • Nguyen Ba, Alex N.; Pogoutse, Anastassia; Provart, Nicholas
  • BMC Bioinformatics, Vol. 10, Issue 1
  • DOI: 10.1186/1471-2105-10-202

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import
journal, July 2001

  • Catimel, Bruno; Teh, Trazel; Fontes, Marcos R. M.
  • Journal of Biological Chemistry, Vol. 276, Issue 36
  • DOI: 10.1074/jbc.M103531200

Structural Basis for the Specificity of Bipartite Nuclear Localization Sequence Binding by Importin-α
journal, April 2003

  • Fontes, Marcos R. M.; Teh, Trazel; Jans, David
  • Journal of Biological Chemistry, Vol. 278, Issue 30
  • DOI: 10.1074/jbc.M303275200

Evolution of the Metazoan-Specific Importin α Gene Family
journal, March 2009

  • Mason, D. Adam; Stage, Deborah E.; Goldfarb, David S.
  • Journal of Molecular Evolution, Vol. 68, Issue 4
  • DOI: 10.1007/s00239-009-9215-8

Energetics by NMR: Site-specific binding in a positively cooperative system
journal, February 2002

  • Tochtrop, G. P.; Richter, K.; Tang, C.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 4
  • DOI: 10.1073/pnas.012379199

TBX5 nuclear localization is mediated by dual cooperative intramolecular signals
journal, October 2003


Cooperative Nuclear Localization Sequences Lend a Novel Role to the N-Terminal Region of MSH6
journal, March 2011


The Homeodomain of Nkx2.2 Carries Two Cooperatively Acting Nuclear Localization Signals
journal, April 2000

  • Hessabi, Behnam; Schmidt, Ines; Walther, Reinhard
  • Biochemical and Biophysical Research Communications, Vol. 270, Issue 3
  • DOI: 10.1006/bbrc.2000.2491

The Krüppel traffic report: Cooperative signals direct KLF8 nuclear transport
journal, September 2009

  • Rodríguez, Estefanía; Martignetti, John A.
  • Cell Research, Vol. 19, Issue 9
  • DOI: 10.1038/cr.2009.103

Hop-on hop-off: importin-α-guided tours to the nucleus in innate immune signaling
journal, January 2013

  • Wirthmueller, Lennart; Roth, Charlotte; Banfield, Mark J.
  • Frontiers in Plant Science, Vol. 4
  • DOI: 10.3389/fpls.2013.00149

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins *
journal, July 1967


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

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

Requirement for theXrcc1DNA Base Excision Repair Gene during Early Mouse Development
journal, April 1999

  • Tebbs, Robert S.; Flannery, Margaret L.; Meneses, Juanito J.
  • Developmental Biology, Vol. 208, Issue 2
  • DOI: 10.1006/dbio.1999.9232

    Works referencing / citing this record:

    Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F
    journal, July 2017


    Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F
    journal, July 2017