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.Screamer3mice exhibit reduced frequencies of peripheral B cells. Flow cytometric analysis of peripheral blood was utilized to determine B cell frequency. 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.Screamer3mice exhibit increased frequencies of peripheral B1b cells. Flow cytometric analysis of peripheral blood was utilized to determine B1b cell frequency. 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.Screamer3mice exhibit increased frequencies of peripheral neutrophils. Flow cytometric analysis of peripheral blood was utilized to determine neutrophil cell frequency. 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 screamer3 phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R0137, some of which showed a reduced frequency of B cells (Figure 1), an increased frequency in B1b cells (Figure 2), and an increased frequency of neutrophils (Figure 3), all in the peripheral blood.
Nature of Mutation
Figure 4.Linkage mapping of the reduced B cell frequency using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 93 mutations (X-axis) identified in the G1 male of pedigree R0137. 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 93 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 15,740,332 (v38) on chromosome 16, or base pair 102,890 in the GenBank genomic region NC_000082 within the donor splice site of intron 51. The strongest association was found with a recessive model of linkage to the reduced frequency of B cells, wherein one variant homozygote departed phenotypically from five homozygous reference mice and three heterozygous mice with a P value of 4.771 x 10-6 (Figure 4).
The effect of the mutation at the cDNA and protein level have not examined, but the mutation is predicted to result in skipping of the 132-nucleotide exon 51 (out of 86 total exons), resulting in an in-frame deletion of 44 amino acids beginning after amino acid 2,250 of the protein, which is normally 4128 amino acids long.
Genomic numbering corresponds to NC_000082. The donor splice site of intron 51, which is destroyed by the screamer3 mutation, is indicated in blue lettering and 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 screamer3 mutation is indcated. 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 constitutes the catalytic domain, designated the PIKK domain, of DNA-PKCS (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 screamer3 mutation affects the splice donor site within intron 51, and is predicted to cause an in-frame deletion of exon 51, which encodes amino acids 2,250 to 2,295. The region deleted in screamer3 is within an undefined portion of DNA-PKCS between TPR3 and the caspase cleavage site.
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 phenotype of the screamer3 mice indicates a loss of function in DNA-PKCSscreamer3.