Impaired telomerase activity in human cells expressing GFP-Ku86 fusion proteins.
The Ku heterodimer is a DNA end-binding protein that promotes the non-homologous end joining (NHEJ) pathway of DNA double strand break (DSB) repair by recruiting the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Ku is also a normal component of telomeres where it is required for telomere maintenance, interacting not only with the DNA but also with various telomere proteins including telomerase. The way in which Ku simultaneously plays such distinct roles, end-joining at DSBs and end-maintenance at telomeres, is unclear. One way to address this is to study cells in which the NHEJ and telomeric roles of Ku have been separated. Here we describe human cells that express fusions between the large human Ku subunit (Ku86) and a fluorescent protein tag. These cells have reduced telomerase activity and increased sensitivity to ionizing radiation (IR) but no change in their DNA-PK activity or in the DNA end-binding of endogenous Ku. Cells with particularly large amounts of one fusion protein undergo progressive telomere shortening and cellular senescence. These data are consistent with models in which Ku recruits telomerase to telomeres or activates recruited telomerase and suggest that the Ku86 fusion proteins specifically block this role.
Badie
C
C
Gene Targeting Group, MRC Clinical Sciences Centre, Imperial College, Hammersmith Hospital Campus, London, UK.
Yáñez-Muñoz
R J
RJ
Muller
C
C
Salles
B
B
Porter
A C G
AC
D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000528:radiation effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D016922:Cellular Senescence; D003001:Cloning, Molecular; D017931:DNA Primers; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005354:Fibrosarcoma; D005784:Gene Amplification; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D016133:Polymerase Chain Reaction; D011839:Radiation, Ionizing; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000528:radiation effects; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D034961:Antigens, Nuclear; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D051747:DNA-Activated Protein Kinase; D019098:Telomerase; D001616:beta-Galactosidase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen