Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.

Abstract

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...

Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells

“…This mRNA, thought to be produced through an alternative splicing event, lacks a 93 bp exon in the PI kinase domain (Blunt et al, 1996;Connelly et al, 1996). Although it might be assumed that this shorter transcript is non-functional, as it deletes 31 amino acids from the conserved region of the PI 3-kinase superfamily, Blunt et al (1996) suggested that it may have a regulatory function. This possibility is strengthened by our ®ndings reported here.…”

“…In spite of the very large size of its transcript, only one alternately spliced mRNA product has been identi®ed. This smaller transcript lacks a small exon encoding a 31 amino acid segment from the amino-terminus of the kinase domain (Blunt et al, 1996;Connelly et al, 1996). Absence of DNA-PK activity confers a radiosensitive phenotype (LeesMiller et al, 1995;Blunt et al, 1996) and as radiosensitivity would be a useful characteristic to induce in malignant cells, an understanding of the mechanisms that control expression of this gene may ultimately lead to a new target in cancer therapy.…”

“…Although the mature 13.5 kb DNA-PK cs mRNA is assembled from approximately 90 small exons by splicing to remove several hundred kb of intron transcript, only one alternatively spliced mRNA has been described (Blunt et al, 1996;Connelly et al, 1996). The smaller transcript, which lacks a 93 nt exon in the kinase domain, was identi®ed as a splicing variant found at a low level (*5%) in cell lines with di erent origins, including epithelial cells, ®broblasts and lymphocytes (Blunt et al, 1996;Connelly et al, 1996).…”

“…The smaller transcript, which lacks a 93 nt exon in the kinase domain, was identi®ed as a splicing variant found at a low level (*5%) in cell lines with di erent origins, including epithelial cells, ®broblasts and lymphocytes (Blunt et al, 1996;Connelly et al, 1996). Since alternate splicing is often tissue speci®c and can have functional and regulatory signi®cance, we checked several of our glioma cell lines to ascertain which forms of the transcript was present in these cells.…”

“…Blunt et al (1996) suggested that the sequence GTTAGC, which is similar to the sequence GGTTAGG at the beginning of this exon, may act as a splice donor site, thereby causing the exon to be omitted from some transcripts. In mouse, the corresponding sequence, GATTAGG, is signi®cantly less similar to the splice donor consensus, and in mouse cells the alternately spliced product is absent.…”