title: An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation.
abstract: The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.
authors: Laboratory of Chromosome and Cell Biology, Rockefeller University, New York, NY, USA. lisa.postow@nih.gov
authors: Lisa
authors: L
authors: Postow
authors: Hironori
authors: H
authors: Funabiki
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D054875:Ubiquitination; D029867:Xenopus Proteins / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015053:Zygote / Q000737:chemistry
chemical_list: D034961:Antigens, Nuclear; C100318:Cullin 1; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; D044786:S-Phase Kinase-Associated Proteins; D029867:Xenopus Proteins; D000072200:Ku Autoantigen
keywords: DNA damage; Fbl12; Fbxl12; Ku70; Ku80; Ku86; SCF; double-strand break; nonhomologous end joining; ubiquitin