Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.

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 identifed 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 carboxylterminal 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.In 1983 Bosma et al.(1) observed a severe combined immunodeficient (scid) mouse in a litter of otherwise normal mice and subsequent backcrossing established the now well-known scid mouse. Only now are details of the molecular defect in scid mice beginning to emerge. scid mice and cell lines derived from them have an interesting and pleiotropic phenotype, with features certainly unpredicted in 1983 (2). scid mice fail to develop mature T and B lymphocytes due to an inability to carry out functional rearrangements of the elements encoding the immunoglobulin and T-cell receptor genes (3-6). These elements, termed the variable (V), diversity (D), and joining (J) segments, are spatially separated in germ-line cells but are rearranged into a contiguous unit during maturation of T and B cells in the process called V(D)J rearrangement (for reviews, see refs. 7-9). This process is initiated by double-strand breaks (dsbs) introduced between partially conserved recombination signal sequences (RSS) and the flanking sequence encoding the V, D, or J element (coding sequence). The rearrangement yields one junction in which the two RSS sequences are rejoined precisely and a second in which the two coding elements are rejoined. The latter junctions invariably harbor small deletions and insertions. scid cells manifest a SCID phenotype due to an inability to form functional coding junctions. In contrast, signal junctions are formed at near normal levels (3-6).In lower organisms, many mutants defective in processes of genetic recombination are sensitive to ionizing radiation (10). This link prompted an examination of scid cell lines, which proved to be both radiosensitive and defective in their ability to rejoin DNA dsbs (11)(12)(13). A number of radiosensitive dsb-repair-defective hamster cell mutants have al...