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.Daffy mice exhibit increased body weights compared to wild-type littermates. Scaled weights 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.Daffy mice exhibit increased CD44 expression on peripheral blood CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 mean fluorescence intensity. 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 daffy phenotype was identified among G3 mice of the pedigree R6000, some of which showed increased body weights compared to wild-type littermates (Figure 1). Some mice also showed increased CD44 expression on peripheral blood CD4 T cells (Figure 2).
Nature of Mutation
Figure 3.Linkage mapping of the increased body weights using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 67 mutations (X-axis) identified in the G1 male of pedigree R6000. Weight 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 67 mutations. Both of the above anomalies were linked to mutations in two genes on chromosome 16: Prkdc and Lamp3. The mutation in Prkdc was presumed causative as the daffy phenotypes mimic that of other Prkdc mutant alleles (see MGI). The Prkdc mutation is an A to T transversion at base pair 15,829,697 (v38) on chromosome 16, or base pair 192,255 in the GenBank genomic region NC_000082 for Prkdc. The strongest association was found with a recessive model of inheritance to the body weight phenotype, wherein two variant homozygotes departed phenotypically from nine homozygous reference mice and 11 heterozygotes with a P value of 0.000211 (Figure 3). A semidominant effect was observed in the CD44+ CD4 mean fluorescence intensity assay.
The mutation corresponds to residue 11,007 in the mRNA sequence NM_011159 within exon 77 of 86 total exons.
The mutated nucleotide is indicated in red. The mutation results in an isoleucine to phenylalanine substitution at position 3,662 (I3662F) in the DNA-PKCS protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.861).
Illustration of Mutations in
Gene & Protein
Figure 4. 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 daffy 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 4). 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 daffy mutation results in an isoleucine to phenylalanine substitution at position 3,662 (I3662F); I3662 is within an undefined domain between the FAT/FATC domain and the PIKK domain.
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 in the daffy mice indicate a loss of DNA-PKCS function.