Characterization of two DNA double-stranded break repair-deficient cell lines that express inactive DNA-dependent protein kinase catalytic subunits.
The DNA-dependent protein kinase (DNA-PK) is a trimeric enzyme consisting of a 460-kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex called Ku, which is comprised of 70 (Ku70) and 86 (Ku80) kDa subunits. Mutations that affect the expression of the catalytic or Ku80 subunits of DNA-PK disrupt both V(D)J recombination and DNA double-stranded break repair pathways. In this report, we show that two previously uncharacterized rodent cell lines that are defective in DNA double-stranded break repair express catalytically inactive DNA-PK. The DNA-PKcs from the DNA double-stranded break repair mutant cell lines IRS-20 and SX-9 assembles on double-stranded DNA but fails to function as a protein kinase. In addition to the kinase defect, the abundance of the DNA-PKcs from both of these cell lines is reduced relative to wild-type controls. These results suggest that the DNA-PKcs gene from each of these cell lines contains mutations that inactivate the enzymatic activity and the expression or stability of the gene product. These data further strengthen the hypothesis that DNA-PK-mediated protein phosphorylation is a necessary component of the DNA double-stranded break repair pathway.
Peterson
S R
SR
Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. spete@telomer.lanl.gov
Stackhouse
M
M
Waltman
M J
MJ
Chen
F
F
Sato
K
K
Chen
D J
DJ
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D008970:Molecular Weight; D016296:Mutagenesis; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D051716:Replication Protein A
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D051716:Replication Protein A; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases