An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.
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.
['Hammel|Michal|M|', 'Yu|Yaping|Y|', 'Radhakrishnan|Sarvan K|SK|', 'Chokshi|Chirayu|C|', 'Tsai|Miaw-Sheue|MS|', 'Matsumoto|Yoshihiro|Y|', 'Kuzdovich|Monica|M|', 'Remesh|Soumya G|SG|', 'Fang|Shujuan|S|', 'Tomkinson|Alan E|AE|', 'Lees-Miller|Susan P|SP|0000-0001-5809-2516', 'Tainer|John A|JA|']
D015153:Blotting, Western D003432:Cross-Linking Reagents D053903:DNA Breaks, Double-Stranded D059766:DNA End-Joining Repair D000072481:DNA Ligase ATP D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase D051747:DNA-Activated Protein Kinase D004268:DNA-Binding Proteins D006367:HeLa Cells D006801:Humans D047468:Immunoprecipitation D000072200:Ku Autoantigen D008958:Models, Molecular D009687:Nuclear Proteins D010766:Phosphorylation D000075223:Poly-ADP-Ribose Binding Proteins D011485:Protein Binding D011487:Protein Conformation D053838:Scattering, Small Angle D014961:X-Ray Diffraction
APLF Ku non-homologous end joining XRCC4 intrinsically disordered protein DNA ligase IV DNA repair DNA-dependent serine/threonine protein kinase (DNA-PK) protein complex small-angle X-ray scattering (SAXS)