DNA-PKcs, Allostery, and DNA Double-Strand Break Repair: Defining the Structure and Setting the Stage.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is central to the regulation of the DNA damage response and repair through nonhomologous end joining. The structure has proved challenging due to its large size and multiple HEAT repeats. We have recently reported crystals of selenomethionine-labeled DNA-PKcs complexed with native KU80ct194 (KU80 residues 539-732) diffracting to 4.3Å resolution. The novel use of crystals of selenomethionine-labeled protein expressed in HeLa cells has facilitated the use of single anomalous X-ray scattering of this 4128 amino acid, multiple HEAT-repeat structure. The monitoring of the selenomethionines in the anomalous-difference density map has allowed the checking of the amino acid residue registration in the electron density, and the labeling of the Ku-C-terminal moiety with selenomethionine has further allowed its identification in the structure of the complex with DNA-PKcs. The crystal structure defines a stage on which many of the components assemble and regulate the kinase activity through modulating the conformation and allosteric regulation of kinase activity.

[1]  B. L. Sibanda,et al.  Crystal structure of an Xrcc4–DNA ligase IV complex , 2001, Nature Structural Biology.

[2]  Laurence H Pearl,et al.  Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). , 2005, Structure.

[3]  David S. Wishart,et al.  VADAR: a web server for quantitative evaluation of protein structure quality , 2003, Nucleic Acids Res..

[4]  Alicia P. Higueruelo,et al.  Arpeggio: A Web Server for Calculating and Visualising Interatomic Interactions in Protein Structures , 2017, Journal of molecular biology.

[5]  T. Blundell,et al.  Structure of the Catalytic Region of DNA Ligase IV in Complex with an Artemis Fragment Sheds Light on Double-Strand Break Repair , 2013, Structure.

[6]  Phoebe L Stewart,et al.  CryoEM and image sorting for flexible protein/DNA complexes. , 2014, Journal of structural biology.

[7]  F. Crick,et al.  The structure of DNA. , 1953, Cold Spring Harbor symposia on quantitative biology.

[8]  A. Fischer,et al.  Cernunnos, a Novel Nonhomologous End-Joining Factor, Is Mutated in Human Immunodeficiency with Microcephaly , 2006, Cell.

[9]  P. Jeggo,et al.  The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit , 1999, Molecular and Cellular Biology.

[10]  David J. Chen,et al.  The Ku80 Carboxy Terminus Stimulates Joining and Artemis-Mediated Processing of DNA Ends , 2008, Molecular and Cellular Biology.

[11]  S. Jackson,et al.  DNA end-joining: from yeast to man. , 1998, Trends in biochemical sciences.

[12]  David F. Burke,et al.  Andante: reducing side-chain rotamer search space during comparative modeling using environment-specific substitution probabilities , 2007, Bioinform..

[13]  Martin Pelikan,et al.  Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex* , 2009, The Journal of Biological Chemistry.

[14]  J. Walker,et al.  Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair , 2001, Nature.

[15]  Penny A. Jeggo,et al.  The role of double-strand break repair — insights from human genetics , 2006, Nature Reviews Genetics.

[16]  B. L. Sibanda,et al.  Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ , 2007, The EMBO journal.

[17]  Laurence H Pearl,et al.  Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. , 2006, Molecular cell.

[18]  Tom L. Blundell,et al.  DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair , 2017, Science.

[19]  T. Blundell,et al.  Achieving high signal-to-noise in cell regulatory systems: Spatial organization of multiprotein transmembrane assemblies of FGFR and MET receptors , 2015, Progress in biophysics and molecular biology.

[20]  M. Lieber,et al.  Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells , 1997, Nature.

[21]  Tom L. Blundell,et al.  PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair , 2015, Science.

[22]  Phoebe L Stewart,et al.  Cryo-EM structure of the DNA-dependent protein kinase catalytic subunit at subnanometer resolution reveals alpha helices and insight into DNA binding. , 2008, Structure.

[23]  W. Hendrickson,et al.  Multi-crystal anomalous diffraction for low-resolution macromolecular phasing. , 2011, Acta crystallographica. Section D, Biological crystallography.

[24]  M. Lieber,et al.  The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. , 2010, Annual review of biochemistry.

[25]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[26]  R. Kornberg,et al.  Structure of DNA‐dependent protein kinase: implications for its regulation by DNA , 1999, The EMBO journal.

[27]  A. Fischer,et al.  Artemis, a Novel DNA Double-Strand Break Repair/V(D)J Recombination Protein, Is Mutated in Human Severe Combined Immune Deficiency , 2001, Cell.

[28]  T. Blundell,et al.  Structural Insights into the Role of Domain Flexibility in Human DNA Ligase IV , 2012, Structure.

[29]  B. L. Sibanda,et al.  Crystal Structure of DNA-PKcs Reveals a Large Open-Ring Cradle Comprised of HEAT Repeats , 2009, Nature.

[30]  John A. Tainer,et al.  XRCC4 Protein Interactions with XRCC4-like Factor (XLF) Create an Extended Grooved Scaffold for DNA Ligation and Double Strand Break Repair , 2011, The Journal of Biological Chemistry.

[31]  J. Widom,et al.  Producing selenomethionine-labeled proteins with a baculovirus expression vector system. , 1999, Structure.

[32]  P L Stewart,et al.  Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase. , 1998, Journal of molecular biology.

[33]  B. L. Sibanda,et al.  Spatial and temporal organization of multi-protein assemblies: achieving sensitive control in information-rich cell-regulatory systems , 2012, Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences.

[34]  B. L. Sibanda,et al.  Structural Biology of DNA Repair: Spatial Organisation of the Multicomponent Complexes of Nonhomologous End Joining , 2010, Journal of nucleic acids.

[35]  S. Lees-Miller,et al.  N-terminal constraint activates the catalytic subunit of the DNA-dependent protein kinase in the absence of DNA or Ku , 2011, Nucleic acids research.

[36]  B. L. Sibanda,et al.  Protein-Protein Interactions in Receptor Activation and Intracellular Signalling , 2000, Biological chemistry.

[37]  Qi Ding,et al.  The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain , 2006, Molecular and Cellular Biology.

[38]  D. Schatz.  V(D)J recombination , 2002, Immunological reviews.

[39]  David J. Chen,et al.  Cell Cycle Dependence of DNA-dependent Protein Kinase Phosphorylation in Response to DNA Double Strand Breaks* , 2005, Journal of Biological Chemistry.

[40]  Qian Wu,et al.  Non-homologous end-joining partners in a helical dance: structural studies of XLF-XRCC4 interactions. , 2011, Biochemical Society transactions.

[41]  S. Jackson,et al.  Mapping of protein-protein interactions within the DNA-dependent protein kinase complex. , 1999, Nucleic acids research.