title: APLF promotes the assembly and activity of non-homologous end joining protein complexes.
abstract: Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, UK.
authors.forename: Gabrielle J
authors.initials: GJ
authors.lastname: Grundy
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Rulten
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zeng
authors.forename: Raquel
authors.initials: R
authors.lastname: Arribas-Bosacoma
authors.forename: Natasha
authors.initials: N
authors.lastname: Iles
authors.forename: Katie
authors.initials: K
authors.lastname: Manley
authors.forename: Antony
authors.initials: A
authors.lastname: Oliver
authors.forename: Keith W
authors.initials: KW
authors.lastname: Caldecott
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D059766:DNA End-Joining Repair; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D000072200:Ku Autoantigen; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D014466:Ultraviolet Rays
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D046912:Multiprotein Complexes; D000075223:Poly-ADP-Ribose Binding Proteins; D011993:Recombinant Fusion Proteins; C101467:XRCC4 protein, human; D004247:DNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; C520557:APLF protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases; D000072481:DNA Ligase ATP