Ku80 removal from DNA through double strand break-induced ubiquitylation.
The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1-Cul1-F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.
Postow
Lisa
L
Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, NY 10065, USA. PostowL@rockefeller.edu
Ghenoiu
Cristina
C
Woo
Eileen M
EM
Krutchinsky
Andrew N
AN
Chait
Brian T
BT
Funabiki
Hironori
H
D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D000072200:Ku Autoantigen; D008954:Models, Biological; D009841:Oligonucleotides / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011995:Recombination, Genetic / Q000235:genetics; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D054875:Ubiquitination; D014981:Xenopus
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D025821:Polyubiquitin; D004247:DNA; D044843:SKP Cullin F-Box Protein Ligases; D046988:Proteasome Endopeptidase Complex; D000072200:Ku Autoantigen