Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products.
Ku, a heterodimer of 70- and 86-kDa subunits, serves as the DNA binding component of the DNA-dependent protein kinase (DNA-PK). Cells deficient for the 86-kDa subunit of Ku (Ku86-deficient cells) lack Ku DNA end-binding activity and are severely defective for formation of the standard V(D)J recombination products, i.e., signal and coding joints. It has been widely hypothesized that Ku is required for protection of broken DNA ends generated during V(D)J recombination. Here we report the first analysis of V(D)J recombination intermediates in a Ku-deficient cell line. We find that full-length, ligatable signal ends are abundant in these cells. These data show that Ku86 is not required for the protection or stabilization of signal ends, suggesting that other proteins may perform this function. The presence of high levels of signal ends in Ku-deficient cells prompted us to investigate whether these ends could participate in joining reactions. We show that nonstandard V(D)J recombination products (hybrid joints), which involve joining a signal end to a coding end, form with similar efficiencies in Ku-deficient and wild-type fibroblasts. These data support the surprising conclusion that Ku is not required for some types of V(D)J joining events. We propose a novel RAG-mediated joining mechanism, analogous to disintegration reactions performed by retroviral integrases, to explain how formation of hybrid joints can bypass the requirement for Ku and DNA-PK.
Han
J O
JO
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Steen
S B
SB
Roth
D B
DB
D016475:3T3 Cells; D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D000072200:Ku Autoantigen; D051379:Mice; D008954:Models, Biological; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D021382:Protein Sorting Signals; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606512:Xrcc5 protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen