title: An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.
abstract: DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.
authors.affiliation: From the Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720, mhammel@lbl.gov.
authors.forename: Michal
authors.initials: M
authors.lastname: Hammel
authors.affiliation: the Department of Biochemistry and Molecular Biology, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Yu
authors.affiliation: the Department of Biochemistry and Molecular Biology, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Sarvan K
authors.initials: SK
authors.lastname: Radhakrishnan
authors.affiliation: From the Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Chirayu
authors.initials: C
authors.lastname: Chokshi
authors.affiliation: From the Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Miaw-Sheue
authors.initials: MS
authors.lastname: Tsai
authors.affiliation: the University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131, and.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Matsumoto
authors.affiliation: From the Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Monica
authors.initials: M
authors.lastname: Kuzdovich
authors.affiliation: From the Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Soumya G
authors.initials: SG
authors.lastname: Remesh
authors.affiliation: the Department of Biochemistry and Molecular Biology, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Fang
authors.affiliation: the University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131, and.
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
authors.affiliation: the Department of Biochemistry and Molecular Biology, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada, leesmill@ucalgary.ca.
authors.forename: Susan P
authors.identifier: 0000-0001-5809-2516
authors.initials: SP
authors.lastname: Lees-Miller
authors.affiliation: From the Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720, JATainer@LBL.gov.|the Department of Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
mesh_terms: D015153:Blotting, Western; D003432:Cross-Linking Reagents; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics*; D000072481:DNA Ligase ATP / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D000072200:Ku Autoantigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D011487:Protein Conformation; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D009687:Nuclear Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen; C520557:APLF protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D000072481:DNA Ligase ATP
keywords: APLF; DNA ligase IV; DNA repair; DNA-dependent serine/threonine protein kinase (DNA-PK); Ku; XRCC4; intrinsically disordered protein; non-homologous end joining; protein complex; small-angle X-ray scattering (SAXS)