title: APLF (C2orf13) facilitates nonhomologous end-joining and undergoes ATM-dependent hyperphosphorylation following ionizing radiation.
abstract: Nonhomologous end-joining (NHEJ) is the major mammalian DNA double-strand break (DSB) repair pathway of DSBs induced by DNA damaging agents. NHEJ is initiated by the recognition of DSBs by the DNA end-binding heterodimer, Ku, and the final step of DNA end-joining is accomplished by the XRCC4-DNA ligase IV complex. We demonstrate that Aprataxin and PNK-like factor (APLF), an endo/exonuclease with an FHA domain and unique zinc fingers (ZFs), interacts with both Ku and XRCC4-DNA ligase IV in human cells. The interaction of APLF with XRCC4-DNA ligase IV is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. In contrast, APLF associates with Ku independently of the FHA and ZF domains, and APLF complexes with Ku at DNA ends. APLF undergoes ionizing radiation (IR) induced ATM-dependent hyperphosphorylation at serine residue 116, which is highly conserved across mammalian APLF homologues. We demonstrate further that depletion of APLF in human cells by siRNA is associated with impaired NHEJ. Collectively, these results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and XRCC4-DNA ligase IV.
authors: Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.
authors: Chloe J
authors: CJ
authors: Macrae
authors: Richard D
authors: RD
authors: McCulloch
authors: Jarkko
authors: J
authors: Ylanko
authors: Daniel
authors: D
authors: Durocher
authors: C Anne
authors: CA
authors: Koch
mesh_terms: D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D000072200:Ku Autoantigen; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D000075223:Poly-ADP-Ribose Binding Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D000075223:Poly-ADP-Ribose Binding Proteins; D025521:Tumor Suppressor Proteins; C101467:XRCC4 protein, human; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen; C520557:APLF protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase