FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes the catalytic subunit of the DNA-dependent protein kinase (DNA-PK). It functions with the Ku70/Ku80 heterodimer protein in DNA double strand break repair and recombination. The protein encoded is a member of the PI3/PI4-kinase family.[provided by RefSeq, Jul 2010] PHENOTYPE: Mutations at this locus effect genome stability, radiation sensitivity and DNA repair. Nonsense (scid) and null homozygotes have severe combined immunodeficiency. A BALB/c variant allele reduces enzyme activity and predisposes to breast cancer. [provided by MGI curators]
Figure 1.Homozygous screamer2mice exhibit diminished T-dependent antibody responses to ovalbumin (OVA) administered with aluminum hydroxide. IgG levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 2.Homozygous screamer2mice exhibit diminished T-dependent antibody responses to recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal). IgG levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 3.Homozygous screamer2mice exhibit diminished T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll). IgM levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
The screamer2 phenotype among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R2852, some of which showed a diminished T-dependent antibody response to ovalbumin (OVA) administered with aluminum hydroxide (Figure 1) as well as a diminished T-dependent antibody response to recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal) (Figure 2). The T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll) was also reduced (Figure 3).
Nature of Mutation
Figure 4.Linkage mapping of the reduced T-independent antibody response to NP-Ficoll using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 45 mutations (X-axis) identified in the G1 male of pedigree R2852. Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.
Whole exome HiSeq sequencing of the G1 grandsire identified 45 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Prkdc: a T to C transition at base pair 15,652,552 (v38) on chromosome 16, or base pair 15,109 in the NC_000082 GenBank genomic region. The strongest association was found with a recessive model of linkage to the normalized T-independent antibody response to NP-Ficoll(P = 1.24 x 10-14), wherein 5 variant homozygotes departed phenotypically from 6 homozygous reference mice and 7 heterozygous mice (Figure 4).
The mutation is located within the donor splice site of intron 7, two nucleotides from the previous exon. Prkdc contains 86 total exons. The effect of the mutation at the cDNA and protein level is unknown. One possibility, shown below, is that aberrant splicing may result in skipping of the 100 base pair exon 7 and out-of-frame splicing from exon 6 to exon 8. This would result in deletion of amino acids 208-241 of the DNA-PKCS protein, coding of 21 aberrant amino acids (amino acids 208-228), and coding of a premature stop codon after amino acid 228.
The donor splice site of intron 7, which is destroyed by the mutation, is indicated in blue; the mutated nucleotide is indicated in red.
Figure 5. Domain structure of DNA-PKCS. The DNA-PKCS protein has three tetratricopeptide repeats (TPR) that are involved in protein-protein interactions such as with the Ku70/Ku80 heterodimer to form the DNA-PK complex. The catalytic site is within the PIKK domain at the C-terminus. FAT domains flank the PI3K/PI4K (PIKK) domain. The N-terminus has three HEAT repeats. The screamer2 mutation is indicated. This image is interactive; click on each mutation to view more information.
The 12,674 base pair Prkdc transcript encodes the 4,128 amino acid, 471 kDa catalytic subunit of the DNA-PK complex, DNA-PKCS. DNA-PKCS is a serine/threonine kinase and member of the phosphatidylinositol 3-kinase-like kinases (PIKK) family (see worker) [reviewed in (1)]. DNA-PKCS has several domains that are essential for its function (Figure 5). A leucine rich region (LRR, aa 1501-1536) mediates the association of DNA-PKCS with C1D, a DNA-binding nuclear matrix-associated factor. The LRR facilitates the intrinsic binding of DNA-PKCS to DNA (2). The DNA-PKCS protein also contains three tetratricopeptide repeats (TPR) (aa 1720-1753, 2921-2954, 2956-2983) that are proposed to assist in protein-protein interactions. A 380 amino acid region at the C-terminus of DNA-PKCS constitutes the catalytic domain, designated the PIKK domain (aa 3747-4015) (3). The PIKK domain is flanked by the FAT domain (named for its homology to FRAP, ATM and TRRAP, aa 2884-3539) and a FATC domain (FAT at the extreme C-terminus, aa 4096-4128) (3). The FAT and FATC domains occur in combination in all PIKK family members. The C-terminal region containing the FATC domain is essential for the kinase activity of DNA-PKCS(4-6). The N-terminal portion of the protein up to the FAT domain consists of HEAT (Huntingtin, Elongation factor 3, A subunit of protein phosphatase 2A and TOR1) repeats (amino acids 288-323, 1001-1037, and 1050-1086) (7). HEAT repeats are helical structural repeats that mediate protein-protein interactions (8).
The screamer2 mutation affects the splice donor site within intron 7, and is predicted to cause skipping of exon 7. A transcript lacking exon 29 would encode the wild type sequence up to amino acid 207, followed by 21 aberrant amino acids and a premature stop. This transcript would likely be subject to nonsense-mediated decay.
Please see the record clover for information about Prkdc.
DNA-PKCS is the catalytic subunit of the DNA-PK complex and is essential for DNA double-strand break repair during nonhomologous end joining (NHEJ) and during the assembly of immune receptor genes (i.e., V(D)J recombination) in developing lymphocytes. Mutations in Prkdc are linked to severe combined immunodeficiency (SCID) in several animal models, a condition marked by lymphopenia, hypogammaglobulinemia, and impaired T and B cell-mediated functions (e.g. defective V(D)J recombination and reduced numbers of peripheral lymphocytes) (9-11). Mutations in Prkdc also exhibit uncapped telomeres and a large number of telomeric fusions, leading to genomic instability (9;12;13). In a spontaneous mouse model of SCID, a DNA-PKCS point mutation resulting in the loss of 83 C-terminal amino acids, a reduction in protein expression, and a block in lymphocyte development has been identified (14). The deficiency in the T-dependent antibody response to OVA-aluminum hydroxide in the screamer2 mice indicates a loss of function in DNA-PKCSscreamer2.
screamer2(F):5'- TCTGGGAGAACTTAAGACCCAG -3'
screamer2(R):5'- ATCCCTCTTAAGTGCATAGCC -3'
screamer2_seq(F):5'- GACCCAGGTATAATACTCCCTATG -3'
screamer2_seq(R):5'- AGCTCCTGGAGACTTGCATTCTG -3'