Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex*

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.

[1]  David J. Chen,et al.  Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. , 2002, Genes & development.

[2]  D. Chan,et al.  The DNA-dependent Protein Kinase Is Inactivated by Autophosphorylation of the Catalytic Subunit (*) , 1996, The Journal of Biological Chemistry.

[3]  M. Jovanović,et al.  Terminal DNA structure and ATP influence binding parameters of the DNA-dependent protein kinase at an early step prior to DNA synapsis , 2006, Nucleic acids research.

[4]  Masao Kakudo,et al.  Small Angle Scattering of X-Rays , 1968 .

[5]  Friedrich Förster,et al.  Integration of small-angle X-ray scattering data into structural modeling of proteins and their assemblies. , 2008, Journal of molecular biology.

[6]  W. Dynan,et al.  Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites , 2002, Molecular and Cellular Biology.

[7]  Claus Müller,et al.  The General Theory , 1998 .

[8]  David J. Chen,et al.  Ku recruits XLF to DNA double‐strand breaks , 2008, EMBO reports.

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

[10]  P. Jeggo,et al.  The impact of a negligent G2/M checkpoint on genomic instability and cancer induction , 2007, Nature Reviews Cancer.

[11]  S. Paillard,et al.  Site‐specific proteolytic cleavage of Ku protein bound to DNA , 1993, Proteins.

[12]  J. Griffith,et al.  Synapsis of DNA ends by DNA‐dependent protein kinase , 2002, The EMBO journal.

[13]  Qi Ding,et al.  Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends. , 2004, Nucleic acids research.

[14]  Qi Ding,et al.  trans Autophosphorylation at DNA-Dependent Protein Kinase's Two Major Autophosphorylation Site Clusters Facilitates End Processing but Not End Joining , 2007, Molecular and Cellular Biology.

[15]  Yunmei Ma,et al.  The DNA-dependent Protein Kinase Catalytic Subunit Phosphorylation Sites in Human Artemis* , 2005, Journal of Biological Chemistry.

[16]  E. Hall,et al.  Radiobiology for the radiologist , 1973 .

[17]  M. Blackledge,et al.  Structural characterization of flexible proteins using small-angle X-ray scattering. , 2007, Journal of the American Chemical Society.

[18]  Stephen P. Jackson,et al.  Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage , 2005, Nature.

[19]  R. Pego,et al.  Analysis of binding reactions by fluorescence recovery after photobleaching. , 2004, Biophysical journal.

[20]  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.

[21]  John A. Tainer,et al.  Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS) , 2009, Nature Methods.

[22]  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.

[23]  John A Tainer,et al.  DNA double-strand break repair from head to tail. , 2002, Current opinion in structural biology.

[24]  John A. Tainer,et al.  Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair , 2009, Cell.

[25]  R. West,et al.  Productive and Nonproductive Complexes of Ku and DNA-Dependent Protein Kinase at DNA Termini , 1998, Molecular and Cellular Biology.

[26]  S. Lees-Miller,et al.  The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation. , 2002, Biochemistry.

[27]  T. Helleday,et al.  DNA double-strand break repair: from mechanistic understanding to cancer treatment. , 2007, DNA repair.

[28]  Katheryn Meek,et al.  Chapter 2 DNA-PK , 2008 .

[29]  David J. Chen,et al.  Solution structure of the C-terminal domain of Ku80 suggests important sites for protein-protein interactions. , 2004, Structure.

[30]  Greg L. Hura,et al.  Structure and flexibility within proteins as identified through small angle X-ray scattering. , 2009, General physiology and biophysics.

[31]  Pablo Chacón,et al.  Visualization of DNA‐induced conformational changes in the DNA repair kinase DNA‐PKcs , 2003, The EMBO journal.

[32]  G. Sapkota,et al.  Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase. , 2002, The Biochemical journal.

[33]  S. Lees-Miller,et al.  Autophosphorylation of DNA-Dependent Protein Kinase Regulates DNA End Processing and May Also Alter Double-Strand Break Repair Pathway Choice , 2005, Molecular and Cellular Biology.

[34]  Y. Hung,et al.  Effect of GAS6 and AXL Gene Polymorphisms on Adiposity, Systemic Inflammation, and Insulin Resistance in Adolescents , 2014, International journal of endocrinology.

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

[36]  K. S. Pawelczak,et al.  A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining , 2008, Nucleic acids research.

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

[38]  F Chen,et al.  The three-dimensional structure of the C-terminal DNA-binding domain of human Ku70. , 2001, The Journal of biological chemistry.

[39]  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.

[40]  GradUate StUdieS,et al.  MOLECULAR AND CELLULAR BIOLOGY , 2009, Molecular and Cellular Biology.

[41]  M. Lieber,et al.  Interaction of DNA‐dependent protein kinase with DNA and with Ku: biochemical and atomic‐force microscopy studies , 1997, The EMBO journal.

[42]  W. Dynan,et al.  Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein. , 1999, Nucleic acids research.

[43]  D I Svergun,et al.  Determination of domain structure of proteins from X-ray solution scattering. , 2001, Biophysical journal.

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

[45]  M. Pelikán,et al.  Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase , 2009, Nucleic acids research.

[46]  David J. Chen,et al.  The endless tale of non-homologous end-joining , 2008, Cell Research.

[47]  Greg L. Hura,et al.  The structure and conformation of Lys63-linked tetraubiquitin. , 2009, Journal of molecular biology.

[48]  Qi Ding,et al.  Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

[50]  D I Svergun,et al.  Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. , 1999, Biophysical journal.

[51]  Timothy Woods,et al.  Autophosphorylation of the Catalytic Subunit of the DNA-Dependent Protein Kinase Is Required for Efficient End Processing during DNA Double-Strand Break Repair , 2003, Molecular and Cellular Biology.

[52]  J. Tainer,et al.  Structure and function of the double-strand break repair machinery. , 2004, DNA Repair.

[53]  John A. Tainer,et al.  X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution , 2007, Quarterly Reviews of Biophysics.

[54]  J Wade Harper,et al.  Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases. , 2009, Molecular cell.

[55]  M. Kerr,et al.  The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling. , 1999, Journal of molecular biology.

[56]  Burkhard Jakob,et al.  Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks , 2007, The Journal of cell biology.

[57]  S. Lees-Miller,et al.  DNA-PK: the means to justify the ends? , 2008, Advances in immunology.

[58]  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.

[59]  良二 上田.  J. Appl. Cryst.の発刊に際して , 1970 .

[60]  L. Pearl,et al.  Structural model of full‐length human Ku70–Ku80 heterodimer and its recognition of DNA and DNA‐PKcs , 2007, EMBO reports.

[61]  Dmitri I. Svergun,et al.  Determination of the regularization parameter in indirect-transform methods using perceptual criteria , 1992 .

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

[63]  S. Lees-Miller,et al.  Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. , 2009, The Biochemical journal.

[64]  Dmitri I Svergun,et al.  Global rigid body modeling of macromolecular complexes against small-angle scattering data. , 2005, Biophysical journal.

[65]  Richard Harris,et al.  The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR). , 2004, Journal of molecular biology.