Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.
DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse.
Blunt
T
T
Medical Research Council Cell Mutation Unit, University of Sussex, Brighton, United Kingdom.
Gell
D
D
Fox
M
M
Taccioli
G E
GE
Lehmann
A R
AR
Jackson
S P
SP
Jeggo
P A
PA
D016475:3T3 Cells; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D019143:Evolution, Molecular; D005091:Exons; D015723:Gene Library; D006801:Humans; D051379:Mice; D016513:Mice, SCID / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics*; D012377:Rodentia; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases