Displacement of DNA-PKcs from DNA ends by the Werner syndrome protein.
The DNA-dependent protein kinase (DNA-PK) complex, which is composed of a DNA-dependent kinase subunit (DNA-PKcs) and the Ku70/80 heterodimer, is involved in DNA double-strand break repair by non-homologous end joining (NHEJ). Ku70/80 interacts with the Werner syndrome protein (WRN) and stimulates WRN exonuclease activity. To investigate a possible function of WRN in NHEJ, we have examined the relationship between DNA-PKcs, Ku and WRN. First, we showed that WRN forms a complex with DNA-PKcs and Ku in solution. Next, we determined whether this complex assembles on DNA ends. Interestingly, the addition of WRN to a Ku:DNA-PKcs:DNA complex results in the displacement of DNA-PKcs from the DNA, indicating that the triple complex WRN:Ku:DNA-PKcs cannot form on DNA ends. The displacement of DNA-PKcs from DNA requires the N- and C-terminal regions of WRN, both of which make direct contact with the Ku70/80 heterodimer. Moreover, exonuclease assays indicate that DNA-PKcs does not protect DNA from the nucleolytic action of WRN. These results suggest that WRN may influence the mechanism by which DNA ends are processed.
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509, Los Angeles, CA 90089, USA.
Baomin
B
Li
Lucio
L
Comai
D034961:Antigens, Nuclear; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D005092:Exonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D005092:Exonucleases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen