Phenotypic Mutation 'clover' (pdf version)
Allele | clover |
Mutation Type |
splice site
(2 bp from exon)
|
Chromosome | 16 |
Coordinate | 15,520,020 bp (GRCm39) |
Base Change | T ⇒ C (forward strand) |
Gene |
Prkdc
|
Gene Name | protein kinase, DNA activated, catalytic polypeptide |
Synonym(s) | slip, DNA-PK, XRCC7, DNAPDcs, DOXNPH, dxnph, DNA-PKcs |
Chromosomal Location |
15,455,730-15,660,099 bp (+) (GRCm39)
|
MGI Phenotype |
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]
|
Accession Number | Ncbi Refseq: NM_011159.2 ; P97313 ; ENSMUST00000023352; MGI:104779
|
Mapped | Yes |
Amino Acid Change |
|
Institutional Source | Beutler Lab |
Gene Model |
not available |
AlphaFold |
no structure available at present |
SMART Domains |
Protein: ENSMUSP00000023352 Gene: ENSMUSG00000022672
Domain | Start | End | E-Value | Type |
low complexity region
|
125 |
138 |
N/A |
INTRINSIC |
low complexity region
|
1253 |
1263 |
N/A |
INTRINSIC |
low complexity region
|
1508 |
1522 |
N/A |
INTRINSIC |
NUC194
|
1810 |
2206 |
2.37e-246 |
SMART |
SCOP:d1gw5a_
|
2210 |
2493 |
5e-3 |
SMART |
low complexity region
|
2669 |
2681 |
N/A |
INTRINSIC |
low complexity region
|
2841 |
2855 |
N/A |
INTRINSIC |
Pfam:FAT
|
3024 |
3470 |
8.2e-75 |
PFAM |
PI3Kc
|
3749 |
4068 |
3.67e-86 |
SMART |
FATC
|
4096 |
4128 |
1.57e-9 |
SMART |
|
Predicted Effect |
probably benign
|
Meta Mutation Damage Score |
Not available |
Is this an essential gene? |
Probably essential (E-score: 0.953) |
Phenotypic Category |
Autosomal Recessive |
Candidate Explorer Status |
loading ... |
Single pedigree Linkage Analysis Data
|
|
Penetrance | |
Alleles Listed at MGI | All alleles(26) : Targeted(4) Gene trapped(16) Transgenic(1) Spontaneous(2) Chemically induced(2) QTL(1)
|
Lab Alleles |
Allele | Source | Chr | Coord | Type | Predicted Effect | PPH Score |
IGL00157:Prkdc
|
APN |
16 |
15515090 |
missense |
probably damaging |
1.00 |
IGL00225:Prkdc
|
APN |
16 |
15627508 |
missense |
possibly damaging |
0.64 |
IGL00481:Prkdc
|
APN |
16 |
15608330 |
missense |
probably benign |
0.41 |
IGL00488:Prkdc
|
APN |
16 |
15593711 |
splice site |
probably null |
|
IGL00489:Prkdc
|
APN |
16 |
15617790 |
missense |
possibly damaging |
0.51 |
IGL00579:Prkdc
|
APN |
16 |
15482103 |
missense |
probably damaging |
1.00 |
IGL00587:Prkdc
|
APN |
16 |
15470222 |
splice site |
probably benign |
|
IGL00666:Prkdc
|
APN |
16 |
15554699 |
missense |
probably damaging |
1.00 |
IGL00675:Prkdc
|
APN |
16 |
15605022 |
missense |
probably benign |
0.05 |
IGL00708:Prkdc
|
APN |
16 |
15597290 |
missense |
probably damaging |
0.97 |
IGL00725:Prkdc
|
APN |
16 |
15634503 |
missense |
probably benign |
0.10 |
IGL00818:Prkdc
|
APN |
16 |
15577618 |
missense |
possibly damaging |
0.92 |
IGL00917:Prkdc
|
APN |
16 |
15557428 |
missense |
probably damaging |
0.98 |
IGL00990:Prkdc
|
APN |
16 |
15519979 |
missense |
probably benign |
0.03 |
IGL01126:Prkdc
|
APN |
16 |
15487185 |
missense |
probably benign |
0.01 |
IGL01141:Prkdc
|
APN |
16 |
15544568 |
missense |
probably damaging |
0.99 |
IGL01306:Prkdc
|
APN |
16 |
15485595 |
missense |
possibly damaging |
0.67 |
IGL01326:Prkdc
|
APN |
16 |
15647556 |
missense |
probably benign |
|
IGL01335:Prkdc
|
APN |
16 |
15634760 |
critical splice donor site |
probably null |
|
IGL01419:Prkdc
|
APN |
16 |
15653030 |
missense |
probably damaging |
1.00 |
IGL01434:Prkdc
|
APN |
16 |
15531451 |
missense |
probably benign |
0.00 |
IGL01554:Prkdc
|
APN |
16 |
15470166 |
missense |
probably benign |
0.05 |
IGL01671:Prkdc
|
APN |
16 |
15485609 |
missense |
possibly damaging |
0.90 |
IGL01871:Prkdc
|
APN |
16 |
15600951 |
missense |
probably benign |
0.00 |
IGL01874:Prkdc
|
APN |
16 |
15552858 |
missense |
possibly damaging |
0.89 |
IGL01930:Prkdc
|
APN |
16 |
15516751 |
missense |
probably damaging |
1.00 |
IGL01984:Prkdc
|
APN |
16 |
15526643 |
missense |
probably benign |
|
IGL02121:Prkdc
|
APN |
16 |
15535048 |
missense |
probably benign |
0.18 |
IGL02152:Prkdc
|
APN |
16 |
15487149 |
missense |
probably benign |
0.15 |
IGL02172:Prkdc
|
APN |
16 |
15627623 |
missense |
probably benign |
0.10 |
IGL02336:Prkdc
|
APN |
16 |
15603843 |
missense |
probably benign |
0.01 |
IGL02336:Prkdc
|
APN |
16 |
15603842 |
missense |
possibly damaging |
0.47 |
IGL02393:Prkdc
|
APN |
16 |
15634622 |
missense |
probably benign |
0.42 |
IGL02406:Prkdc
|
APN |
16 |
15488399 |
missense |
probably benign |
0.00 |
IGL02500:Prkdc
|
APN |
16 |
15532146 |
critical splice donor site |
probably null |
|
IGL02568:Prkdc
|
APN |
16 |
15544406 |
missense |
probably damaging |
0.98 |
IGL02579:Prkdc
|
APN |
16 |
15488465 |
missense |
possibly damaging |
0.83 |
IGL02652:Prkdc
|
APN |
16 |
15600951 |
missense |
probably benign |
0.00 |
IGL02661:Prkdc
|
APN |
16 |
15587689 |
missense |
possibly damaging |
0.92 |
IGL02685:Prkdc
|
APN |
16 |
15653907 |
missense |
possibly damaging |
0.61 |
IGL02741:Prkdc
|
APN |
16 |
15570590 |
splice site |
probably benign |
|
IGL02803:Prkdc
|
APN |
16 |
15651530 |
splice site |
probably benign |
|
IGL02866:Prkdc
|
APN |
16 |
15649191 |
missense |
probably damaging |
1.00 |
IGL02882:Prkdc
|
APN |
16 |
15469383 |
nonsense |
probably null |
|
IGL02989:Prkdc
|
APN |
16 |
15617880 |
missense |
possibly damaging |
0.67 |
IGL03053:Prkdc
|
APN |
16 |
15652030 |
missense |
probably benign |
0.02 |
IGL03071:Prkdc
|
APN |
16 |
15617848 |
missense |
probably benign |
0.01 |
IGL03091:Prkdc
|
APN |
16 |
15523174 |
splice site |
probably benign |
|
IGL03100:Prkdc
|
APN |
16 |
15531499 |
missense |
probably benign |
0.08 |
IGL03128:Prkdc
|
APN |
16 |
15518608 |
splice site |
probably benign |
|
IGL03168:Prkdc
|
APN |
16 |
15652030 |
missense |
probably benign |
0.02 |
IGL03204:Prkdc
|
APN |
16 |
15587665 |
missense |
probably benign |
0.01 |
IGL03390:Prkdc
|
APN |
16 |
15488490 |
nonsense |
probably null |
|
anhimid
|
UTSW |
16 |
15543325 |
critical splice donor site |
probably null |
|
anhinga
|
UTSW |
16 |
15526796 |
critical splice donor site |
probably null |
|
Bushtit
|
UTSW |
16 |
15570628 |
missense |
probably damaging |
0.97 |
crackle
|
UTSW |
16 |
15603914 |
critical splice donor site |
probably null |
|
Daffy
|
UTSW |
16 |
15647561 |
missense |
possibly damaging |
0.86 |
darter
|
UTSW |
16 |
15591477 |
missense |
possibly damaging |
0.93 |
Elmer_fudd
|
UTSW |
16 |
15625922 |
missense |
probably benign |
0.01 |
envenomation
|
UTSW |
16 |
15653091 |
nonsense |
probably null |
|
hobgoblin
|
UTSW |
16 |
15633850 |
missense |
probably damaging |
1.00 |
Incubus
|
UTSW |
16 |
15490191 |
missense |
probably damaging |
1.00 |
liming
|
UTSW |
16 |
15570693 |
nonsense |
probably null |
|
newt
|
UTSW |
16 |
15545590 |
missense |
probably benign |
0.04 |
ornithorhynchus
|
UTSW |
16 |
15634523 |
critical splice donor site |
probably null |
|
primitive
|
UTSW |
16 |
15653022 |
frame shift |
probably null |
|
roadrunner
|
UTSW |
16 |
15651751 |
missense |
probably damaging |
1.00 |
Schreier
|
UTSW |
16 |
15488392 |
missense |
probably benign |
0.00 |
screamer
|
UTSW |
16 |
15649146 |
nonsense |
probably null |
|
Screamer10
|
UTSW |
16 |
15585889 |
missense |
probably damaging |
0.98 |
screamer2
|
UTSW |
16 |
15470416 |
critical splice donor site |
probably null |
|
screamer3
|
UTSW |
16 |
15558196 |
critical splice donor site |
probably null |
|
screamer4
|
UTSW |
16 |
15600943 |
missense |
probably benign |
0.00 |
screamer5
|
UTSW |
16 |
15505268 |
missense |
probably benign |
|
screamer6
|
UTSW |
16 |
15577469 |
missense |
probably damaging |
1.00 |
screamer7
|
UTSW |
16 |
15472681 |
splice site |
probably null |
|
Screamer8
|
UTSW |
16 |
15537297 |
missense |
probably benign |
0.00 |
Screamer9
|
UTSW |
16 |
15552786 |
missense |
probably benign |
0.01 |
Tweetie
|
UTSW |
16 |
15535665 |
missense |
probably damaging |
1.00 |
updock
|
UTSW |
16 |
15612958 |
missense |
probably benign |
|
ANU23:Prkdc
|
UTSW |
16 |
15485595 |
missense |
possibly damaging |
0.67 |
R0008:Prkdc
|
UTSW |
16 |
15526565 |
splice site |
probably benign |
|
R0018:Prkdc
|
UTSW |
16 |
15544406 |
missense |
probably benign |
0.03 |
R0018:Prkdc
|
UTSW |
16 |
15544406 |
missense |
probably benign |
0.03 |
R0069:Prkdc
|
UTSW |
16 |
15544368 |
missense |
probably benign |
0.03 |
R0125:Prkdc
|
UTSW |
16 |
15516871 |
missense |
probably damaging |
0.98 |
R0131:Prkdc
|
UTSW |
16 |
15531517 |
missense |
probably benign |
0.09 |
R0131:Prkdc
|
UTSW |
16 |
15531517 |
missense |
probably benign |
0.09 |
R0132:Prkdc
|
UTSW |
16 |
15531517 |
missense |
probably benign |
0.09 |
R0137:Prkdc
|
UTSW |
16 |
15558196 |
critical splice donor site |
probably null |
|
R0334:Prkdc
|
UTSW |
16 |
15554663 |
missense |
probably benign |
0.00 |
R0373:Prkdc
|
UTSW |
16 |
15609791 |
missense |
probably damaging |
1.00 |
R0485:Prkdc
|
UTSW |
16 |
15651604 |
missense |
probably damaging |
0.97 |
R0511:Prkdc
|
UTSW |
16 |
15649146 |
nonsense |
probably null |
|
R0538:Prkdc
|
UTSW |
16 |
15651652 |
missense |
probably damaging |
1.00 |
R0595:Prkdc
|
UTSW |
16 |
15625952 |
missense |
probably damaging |
1.00 |
R0607:Prkdc
|
UTSW |
16 |
15589921 |
missense |
probably damaging |
0.98 |
R0616:Prkdc
|
UTSW |
16 |
15508271 |
missense |
probably damaging |
1.00 |
R0630:Prkdc
|
UTSW |
16 |
15628665 |
missense |
probably damaging |
1.00 |
R0694:Prkdc
|
UTSW |
16 |
15586501 |
missense |
probably damaging |
1.00 |
R0702:Prkdc
|
UTSW |
16 |
15603835 |
missense |
possibly damaging |
0.95 |
R0965:Prkdc
|
UTSW |
16 |
15647580 |
missense |
probably benign |
|
R1027:Prkdc
|
UTSW |
16 |
15468576 |
missense |
possibly damaging |
0.80 |
R1029:Prkdc
|
UTSW |
16 |
15472613 |
splice site |
probably benign |
|
R1033:Prkdc
|
UTSW |
16 |
15585815 |
missense |
probably damaging |
1.00 |
R1067:Prkdc
|
UTSW |
16 |
15570646 |
missense |
probably damaging |
0.99 |
R1116:Prkdc
|
UTSW |
16 |
15600943 |
missense |
probably benign |
0.00 |
R1187:Prkdc
|
UTSW |
16 |
15577610 |
missense |
probably damaging |
0.98 |
R1226:Prkdc
|
UTSW |
16 |
15491861 |
missense |
possibly damaging |
0.80 |
R1279:Prkdc
|
UTSW |
16 |
15508146 |
missense |
probably damaging |
1.00 |
R1304:Prkdc
|
UTSW |
16 |
15577587 |
missense |
probably damaging |
0.99 |
R1314:Prkdc
|
UTSW |
16 |
15482091 |
missense |
possibly damaging |
0.68 |
R1351:Prkdc
|
UTSW |
16 |
15485564 |
missense |
possibly damaging |
0.62 |
R1509:Prkdc
|
UTSW |
16 |
15549430 |
missense |
probably damaging |
1.00 |
R1512:Prkdc
|
UTSW |
16 |
15505268 |
missense |
probably benign |
|
R1531:Prkdc
|
UTSW |
16 |
15589970 |
missense |
probably benign |
0.01 |
R1579:Prkdc
|
UTSW |
16 |
15493192 |
missense |
probably benign |
0.00 |
R1669:Prkdc
|
UTSW |
16 |
15551922 |
missense |
probably damaging |
1.00 |
R1682:Prkdc
|
UTSW |
16 |
15494853 |
missense |
probably benign |
0.19 |
R1713:Prkdc
|
UTSW |
16 |
15612958 |
missense |
probably benign |
|
R1762:Prkdc
|
UTSW |
16 |
15455825 |
missense |
probably benign |
|
R1789:Prkdc
|
UTSW |
16 |
15557388 |
missense |
probably damaging |
1.00 |
R1822:Prkdc
|
UTSW |
16 |
15577469 |
missense |
probably damaging |
1.00 |
R1848:Prkdc
|
UTSW |
16 |
15625922 |
missense |
probably benign |
0.01 |
R1887:Prkdc
|
UTSW |
16 |
15647499 |
missense |
probably benign |
0.00 |
R1891:Prkdc
|
UTSW |
16 |
15543300 |
missense |
probably benign |
0.02 |
R1921:Prkdc
|
UTSW |
16 |
15532079 |
missense |
possibly damaging |
0.80 |
R1922:Prkdc
|
UTSW |
16 |
15532130 |
missense |
probably benign |
0.00 |
R1929:Prkdc
|
UTSW |
16 |
15472681 |
splice site |
probably null |
|
R1939:Prkdc
|
UTSW |
16 |
15653777 |
missense |
possibly damaging |
0.95 |
R2021:Prkdc
|
UTSW |
16 |
15494873 |
missense |
probably benign |
0.00 |
R2033:Prkdc
|
UTSW |
16 |
15505216 |
splice site |
probably benign |
|
R2056:Prkdc
|
UTSW |
16 |
15545469 |
missense |
probably benign |
0.03 |
R2057:Prkdc
|
UTSW |
16 |
15545469 |
missense |
probably benign |
0.03 |
R2058:Prkdc
|
UTSW |
16 |
15545469 |
missense |
probably benign |
0.03 |
R2082:Prkdc
|
UTSW |
16 |
15533827 |
missense |
probably damaging |
1.00 |
R2109:Prkdc
|
UTSW |
16 |
15505254 |
missense |
probably benign |
0.01 |
R2124:Prkdc
|
UTSW |
16 |
15537297 |
missense |
probably benign |
0.00 |
R2164:Prkdc
|
UTSW |
16 |
15523071 |
missense |
probably damaging |
1.00 |
R2174:Prkdc
|
UTSW |
16 |
15552786 |
missense |
probably benign |
0.01 |
R2191:Prkdc
|
UTSW |
16 |
15516688 |
missense |
probably damaging |
1.00 |
R2270:Prkdc
|
UTSW |
16 |
15472681 |
splice site |
probably null |
|
R2271:Prkdc
|
UTSW |
16 |
15472681 |
splice site |
probably null |
|
R2272:Prkdc
|
UTSW |
16 |
15472681 |
splice site |
probably null |
|
R2356:Prkdc
|
UTSW |
16 |
15502068 |
missense |
probably benign |
|
R2852:Prkdc
|
UTSW |
16 |
15470416 |
critical splice donor site |
probably null |
|
R3115:Prkdc
|
UTSW |
16 |
15482222 |
missense |
probably benign |
0.01 |
R3116:Prkdc
|
UTSW |
16 |
15482222 |
missense |
probably benign |
0.01 |
R3499:Prkdc
|
UTSW |
16 |
15585889 |
missense |
probably damaging |
0.98 |
R3687:Prkdc
|
UTSW |
16 |
15617831 |
missense |
probably benign |
|
R3834:Prkdc
|
UTSW |
16 |
15609810 |
missense |
probably damaging |
1.00 |
R3835:Prkdc
|
UTSW |
16 |
15609810 |
missense |
probably damaging |
1.00 |
R3961:Prkdc
|
UTSW |
16 |
15647475 |
splice site |
probably null |
|
R4151:Prkdc
|
UTSW |
16 |
15634637 |
missense |
probably benign |
|
R4233:Prkdc
|
UTSW |
16 |
15653783 |
missense |
probably benign |
0.11 |
R4281:Prkdc
|
UTSW |
16 |
15623963 |
splice site |
probably null |
|
R4296:Prkdc
|
UTSW |
16 |
15555769 |
missense |
probably damaging |
0.99 |
R4344:Prkdc
|
UTSW |
16 |
15585886 |
missense |
probably damaging |
0.98 |
R4424:Prkdc
|
UTSW |
16 |
15653946 |
missense |
probably damaging |
1.00 |
R4424:Prkdc
|
UTSW |
16 |
15591603 |
missense |
probably damaging |
0.98 |
R4497:Prkdc
|
UTSW |
16 |
15518517 |
missense |
probably benign |
0.43 |
R4549:Prkdc
|
UTSW |
16 |
15554734 |
missense |
possibly damaging |
0.89 |
R4594:Prkdc
|
UTSW |
16 |
15585830 |
missense |
possibly damaging |
0.64 |
R4603:Prkdc
|
UTSW |
16 |
15628688 |
missense |
probably damaging |
0.98 |
R4615:Prkdc
|
UTSW |
16 |
15480938 |
missense |
probably damaging |
0.99 |
R4648:Prkdc
|
UTSW |
16 |
15634638 |
missense |
probably benign |
0.05 |
R4662:Prkdc
|
UTSW |
16 |
15551916 |
missense |
probably damaging |
1.00 |
R4680:Prkdc
|
UTSW |
16 |
15589894 |
missense |
probably benign |
0.00 |
R4700:Prkdc
|
UTSW |
16 |
15519976 |
missense |
probably damaging |
1.00 |
R4716:Prkdc
|
UTSW |
16 |
15628701 |
missense |
probably benign |
0.32 |
R4720:Prkdc
|
UTSW |
16 |
15485579 |
missense |
probably benign |
|
R4785:Prkdc
|
UTSW |
16 |
15466840 |
missense |
probably benign |
0.21 |
R4822:Prkdc
|
UTSW |
16 |
15468576 |
missense |
possibly damaging |
0.80 |
R4829:Prkdc
|
UTSW |
16 |
15519939 |
missense |
possibly damaging |
0.80 |
R4981:Prkdc
|
UTSW |
16 |
15496173 |
missense |
probably damaging |
1.00 |
R4989:Prkdc
|
UTSW |
16 |
15491861 |
missense |
possibly damaging |
0.80 |
R5059:Prkdc
|
UTSW |
16 |
15655882 |
missense |
probably damaging |
1.00 |
R5074:Prkdc
|
UTSW |
16 |
15589912 |
missense |
probably damaging |
1.00 |
R5115:Prkdc
|
UTSW |
16 |
15608444 |
missense |
probably benign |
|
R5151:Prkdc
|
UTSW |
16 |
15533899 |
missense |
probably damaging |
1.00 |
R5165:Prkdc
|
UTSW |
16 |
15496136 |
missense |
probably damaging |
1.00 |
R5215:Prkdc
|
UTSW |
16 |
15589985 |
missense |
possibly damaging |
0.64 |
R5270:Prkdc
|
UTSW |
16 |
15552819 |
missense |
probably damaging |
1.00 |
R5278:Prkdc
|
UTSW |
16 |
15532838 |
missense |
probably damaging |
1.00 |
R5351:Prkdc
|
UTSW |
16 |
15649176 |
missense |
probably benign |
0.03 |
R5416:Prkdc
|
UTSW |
16 |
15623814 |
missense |
probably damaging |
1.00 |
R5418:Prkdc
|
UTSW |
16 |
15612961 |
missense |
probably benign |
0.20 |
R5437:Prkdc
|
UTSW |
16 |
15587739 |
missense |
possibly damaging |
0.46 |
R5452:Prkdc
|
UTSW |
16 |
15586501 |
missense |
possibly damaging |
0.96 |
R5518:Prkdc
|
UTSW |
16 |
15496172 |
missense |
probably damaging |
1.00 |
R5538:Prkdc
|
UTSW |
16 |
15469333 |
missense |
probably damaging |
1.00 |
R5589:Prkdc
|
UTSW |
16 |
15524655 |
missense |
probably benign |
0.02 |
R5618:Prkdc
|
UTSW |
16 |
15627476 |
missense |
probably damaging |
1.00 |
R5640:Prkdc
|
UTSW |
16 |
15647633 |
missense |
possibly damaging |
0.86 |
R5661:Prkdc
|
UTSW |
16 |
15628634 |
missense |
possibly damaging |
0.81 |
R5771:Prkdc
|
UTSW |
16 |
15482097 |
missense |
probably damaging |
1.00 |
R5772:Prkdc
|
UTSW |
16 |
15597252 |
missense |
possibly damaging |
0.49 |
R5783:Prkdc
|
UTSW |
16 |
15535665 |
missense |
probably damaging |
1.00 |
R5792:Prkdc
|
UTSW |
16 |
15634616 |
missense |
probably damaging |
1.00 |
R5797:Prkdc
|
UTSW |
16 |
15555698 |
nonsense |
probably null |
|
R5826:Prkdc
|
UTSW |
16 |
15551962 |
missense |
probably benign |
|
R5883:Prkdc
|
UTSW |
16 |
15533778 |
missense |
probably benign |
|
R5895:Prkdc
|
UTSW |
16 |
15570693 |
nonsense |
probably null |
|
R5998:Prkdc
|
UTSW |
16 |
15601021 |
missense |
probably damaging |
1.00 |
R6000:Prkdc
|
UTSW |
16 |
15647561 |
missense |
possibly damaging |
0.86 |
R6120:Prkdc
|
UTSW |
16 |
15557335 |
missense |
probably benign |
0.00 |
R6145:Prkdc
|
UTSW |
16 |
15589937 |
missense |
probably damaging |
1.00 |
R6209:Prkdc
|
UTSW |
16 |
15608456 |
missense |
probably damaging |
1.00 |
R6293:Prkdc
|
UTSW |
16 |
15605019 |
missense |
probably benign |
0.00 |
R6321:Prkdc
|
UTSW |
16 |
15532783 |
missense |
probably benign |
|
R6376:Prkdc
|
UTSW |
16 |
15587749 |
missense |
probably benign |
0.06 |
R6387:Prkdc
|
UTSW |
16 |
15516679 |
missense |
probably benign |
0.01 |
R6406:Prkdc
|
UTSW |
16 |
15535665 |
missense |
probably damaging |
1.00 |
R6469:Prkdc
|
UTSW |
16 |
15612939 |
missense |
probably benign |
0.10 |
R6486:Prkdc
|
UTSW |
16 |
15570628 |
missense |
probably damaging |
0.97 |
R6665:Prkdc
|
UTSW |
16 |
15603914 |
critical splice donor site |
probably null |
|
R6703:Prkdc
|
UTSW |
16 |
15488392 |
missense |
probably benign |
0.00 |
R6774:Prkdc
|
UTSW |
16 |
15543325 |
critical splice donor site |
probably null |
|
R6854:Prkdc
|
UTSW |
16 |
15469402 |
missense |
probably damaging |
1.00 |
R6878:Prkdc
|
UTSW |
16 |
15594936 |
missense |
probably benign |
0.31 |
R6882:Prkdc
|
UTSW |
16 |
15626020 |
missense |
probably benign |
0.33 |
R6882:Prkdc
|
UTSW |
16 |
15601127 |
critical splice donor site |
probably null |
|
R6949:Prkdc
|
UTSW |
16 |
15617853 |
missense |
probably benign |
|
R6950:Prkdc
|
UTSW |
16 |
15633850 |
missense |
probably damaging |
1.00 |
R7019:Prkdc
|
UTSW |
16 |
15587830 |
missense |
probably benign |
0.00 |
R7064:Prkdc
|
UTSW |
16 |
15608317 |
missense |
probably benign |
0.00 |
R7097:Prkdc
|
UTSW |
16 |
15507207 |
missense |
probably damaging |
1.00 |
R7201:Prkdc
|
UTSW |
16 |
15516667 |
missense |
probably benign |
0.12 |
R7235:Prkdc
|
UTSW |
16 |
15532127 |
missense |
probably benign |
|
R7283:Prkdc
|
UTSW |
16 |
15535628 |
missense |
probably benign |
0.00 |
R7401:Prkdc
|
UTSW |
16 |
15466602 |
missense |
probably damaging |
1.00 |
R7525:Prkdc
|
UTSW |
16 |
15490191 |
missense |
probably damaging |
1.00 |
R7647:Prkdc
|
UTSW |
16 |
15555807 |
missense |
probably damaging |
1.00 |
R7679:Prkdc
|
UTSW |
16 |
15649183 |
missense |
probably damaging |
1.00 |
R7803:Prkdc
|
UTSW |
16 |
15623960 |
missense |
probably null |
0.05 |
R7858:Prkdc
|
UTSW |
16 |
15507141 |
missense |
probably benign |
0.11 |
R7872:Prkdc
|
UTSW |
16 |
15532870 |
missense |
probably benign |
0.05 |
R7896:Prkdc
|
UTSW |
16 |
15526767 |
missense |
probably damaging |
0.97 |
R8032:Prkdc
|
UTSW |
16 |
15597315 |
missense |
probably benign |
0.00 |
R8055:Prkdc
|
UTSW |
16 |
15634749 |
missense |
probably benign |
0.09 |
R8153:Prkdc
|
UTSW |
16 |
15482108 |
missense |
probably damaging |
1.00 |
R8281:Prkdc
|
UTSW |
16 |
15523117 |
missense |
probably damaging |
1.00 |
R8302:Prkdc
|
UTSW |
16 |
15653946 |
missense |
probably damaging |
1.00 |
R8322:Prkdc
|
UTSW |
16 |
15532005 |
splice site |
probably benign |
|
R8401:Prkdc
|
UTSW |
16 |
15591477 |
missense |
possibly damaging |
0.93 |
R8440:Prkdc
|
UTSW |
16 |
15653022 |
frame shift |
probably null |
|
R8458:Prkdc
|
UTSW |
16 |
15608540 |
critical splice donor site |
probably null |
|
R8472:Prkdc
|
UTSW |
16 |
15469400 |
missense |
probably damaging |
1.00 |
R8478:Prkdc
|
UTSW |
16 |
15466788 |
missense |
probably benign |
0.00 |
R8515:Prkdc
|
UTSW |
16 |
15482232 |
missense |
probably damaging |
1.00 |
R8546:Prkdc
|
UTSW |
16 |
15480899 |
missense |
probably damaging |
1.00 |
R8678:Prkdc
|
UTSW |
16 |
15526796 |
critical splice donor site |
probably null |
|
R8739:Prkdc
|
UTSW |
16 |
15626068 |
missense |
probably benign |
0.01 |
R8749:Prkdc
|
UTSW |
16 |
15601029 |
missense |
possibly damaging |
0.85 |
R8836:Prkdc
|
UTSW |
16 |
15545523 |
missense |
probably damaging |
1.00 |
R8904:Prkdc
|
UTSW |
16 |
15545590 |
missense |
probably benign |
0.04 |
R8952:Prkdc
|
UTSW |
16 |
15491624 |
intron |
probably benign |
|
R8971:Prkdc
|
UTSW |
16 |
15493229 |
missense |
probably null |
0.99 |
R8974:Prkdc
|
UTSW |
16 |
15617726 |
splice site |
probably null |
|
R9052:Prkdc
|
UTSW |
16 |
15508160 |
missense |
probably benign |
0.05 |
R9069:Prkdc
|
UTSW |
16 |
15653091 |
nonsense |
probably null |
|
R9200:Prkdc
|
UTSW |
16 |
15523153 |
missense |
probably damaging |
1.00 |
R9235:Prkdc
|
UTSW |
16 |
15651751 |
missense |
probably damaging |
1.00 |
R9278:Prkdc
|
UTSW |
16 |
15634523 |
critical splice donor site |
probably null |
|
R9309:Prkdc
|
UTSW |
16 |
15526792 |
nonsense |
probably null |
|
R9386:Prkdc
|
UTSW |
16 |
15496136 |
missense |
probably damaging |
0.99 |
R9452:Prkdc
|
UTSW |
16 |
15485465 |
missense |
possibly damaging |
0.90 |
R9500:Prkdc
|
UTSW |
16 |
15657079 |
missense |
possibly damaging |
0.76 |
R9608:Prkdc
|
UTSW |
16 |
15548335 |
missense |
probably damaging |
1.00 |
R9608:Prkdc
|
UTSW |
16 |
15548334 |
missense |
possibly damaging |
0.96 |
R9636:Prkdc
|
UTSW |
16 |
15548341 |
missense |
probably benign |
0.19 |
R9656:Prkdc
|
UTSW |
16 |
15617818 |
missense |
probably benign |
0.00 |
R9674:Prkdc
|
UTSW |
16 |
15533819 |
missense |
probably damaging |
0.98 |
R9760:Prkdc
|
UTSW |
16 |
15657044 |
nonsense |
probably null |
|
X0023:Prkdc
|
UTSW |
16 |
15558142 |
missense |
probably benign |
|
Z1176:Prkdc
|
UTSW |
16 |
15505286 |
nonsense |
probably null |
|
|
Mode of Inheritance |
Autosomal Recessive |
Local Stock | Live Mice, Sperm |
MMRRC Submission |
036486-MU
|
Last Updated |
2019-07-17 4:33 PM
by Diantha La Vine
|
Record Created |
2011-02-08 5:40 PM
by Carrie N. Arnold
|
Record Posted |
2012-06-25 |
Phenotypic Description |
The clover mice exhibited a reduced T-independent IgM response to NP-Ficoll, and a null T-dependent IgG response to a vector based on Semliki Forest Virus encoding β-galactosidase (Figure 1). Peripheral lymphocyte frequencies have not been assessed.
|
Nature of Mutation |
The clover mutation was mapped using bulk segregation analysis (BSA) of F2 backcross offspring using C57BL/10J as the mapping strain. The mutation showed strongest linkage with marker B10SNPS0225 at position 29856457 bp on Chromosome 16 (synthetic LOD = 3.6). Whole genome SOLiD sequencing of a homozygous clover mouse and validation by capillary sequencing identified a T to C transition at bp 15702250, 14.2 Mb from the marker of peak linkage, on chromosome 16 in the Genbank genomic region NC_000082. The effect of the mutation at the cDNA and protein level has not been tested.
<--exon 28 <--exon 29 intron 29--> exon 30-->
62863 AAATCCTTAG……TTGCCACA gtaggtgata…………GGGATTTCCACCTTTGA
1116 -K--S--L--……-L--P--Q G--D--F--H--L--* 1124
correct deleted aberrant
|
The mutation is in intron 29, within the donor splice site, 2 nucleotides after exon 29. The mutated nucleotide is indicated in red; the splice donor sequence is shown in blue.
|
Illustration of Mutations in
Gene & Protein |
|
---|
Protein Prediction |
The 12,674 base pair Prkdc transcript encodes the 4,128 amino acid, 471 kDa catalytic subunit of the DNA-PK complex, DNA-PKCS (Figure 2 & 3). DNA-PKCS is a serine/threonine kinase and member of the phosphatidylinositol 3-kinase-like kinases (PIKK) family (see worker) [reviewed in (1)]. The PIKK family is similar to the PI3K family, with the exception that PIKK family members do not phosphorylate lipids [reviewed in (2)], but rather hundreds of proteins involved in the regulation of cell cycle progression, DNA repair, apoptosis, and cellular senescence (3). The PIKK family members ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related), along with DNA-PKCS, are involved in DNA repair [(4); reviewed in (5)]. In particular, DNA-PKCS is required for repair of DNA double strand breaks (DSB) during the process of nonhomologous end joining (NHEJ), and during the assembly of immune receptor genes in developing lymphocytes [V(D)J recombination]. Additional members of the PIKK family are SMG1 homolog (SMG1), mammalian target of rapamycin (mTOR), and transformation/transcription domain-associated protein (TRRAP), which are involved in nonsense-mediated decay of mRNA, regulation of nutrient-dependent signaling, and regulation of chromatin during transcription, respectively (3). DNA-PKCS has several domains that are essential for its function (Figure 2 & 3). A leucine rich region (LRR, aa 1501-1536) mediates the association of DNA-PKCS with C1D, a DNA-binding nuclear matrix-associated factor that may be involved in gene regulation (1;6), and which is involved in NHEJ and homologous recombination in yeast (7). The LRR facilitates the intrinsic binding of DNA-PKCS to DNA (6), which occurs at low (nonphysiologic) salt concentrations and activates kinase activity (8); point mutations in the LRR reduce the ability of DNA-PKCS to rescue defective V(D)J recombination and radiosensitivity in DNA-PKCS-deficient cells (6). Association with C1D may contribute to the weak intrinsic DNA binding activity of DNA-PKCS. 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. TPR2 and TPR3 exist within a larger region that mediates binding of DNA-PKCS to the Ku heterodimer (Ku70 and Ku80), which is required to target DNA-PKCS to broken DNA ends in vivo. TPR1 forms part of a region shown to interact with Lyn tyrosine kinase (9). A 380 amino acid region at the C terminus constitutes the catalytic domain, designated the PIKK domain, of DNA-PKCS (aa 3747-4015) (1;10). 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) (10). The FAT and FATC domains occur in combination in all PIKK family members, suggesting a possible role in maintaining a structural conformation essential for the activation of the catalytic site (1;2). It has been shown the C-terminal region containing the FATC domain is essential for the kinase activity of DNA-PKCS (11-13). 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 (14). HEAT repeats are helical structural repeats that mediate protein-protein interactions (15). In DNA-PKCS they are believed to contain recruitment sites for DNA repair proteins (16), and to hold the DNA in place while it is being repaired. Autophosphorylation occurs on multiple sites across the length of DNA-PKCS and is essential for its function (16-19). Two major clusters of serine/threonine autophosphorylation sites near the center of the protein reciprocally regulate the activity of DNA-PKCS [(20-22); reviewed in (2;23)]. The ABCDE cluster contains six sites spanning resides 2609-2647 and their autophosphorylation is thought to induce a structural change in the protein that facilitates its release from the Ku dimer and the DNA as well as the inactivation of DNA-PKCS kinase activity (1;2;14;23). The result is DNA end processing by repair proteins that are presumably granted access to the broken DNA ends (20;21). The PQR cluster contains five sites spanning residues 2023-2056; phosphorylation of these sites inhibits DNA end processing (24). Site-directed mutational analysis demonstrated that phosphorylation of one or two sites within each cluster is functionally sufficient; only when all five or six sites were mutated was NHEJ impaired. Whether autophosphorylation of DNA-PKCS occurs in cis or in trans remains unknown (25;26); however, the interaction of two DNA-PKCS molecules on adjacent sides of a DSB to form a ‘synaptic complex’ is believed necessary prior to DNA end processing. DNA-PKCS also undergoes autophosphorylation within the kinase activation loop (22). Autophosphorylation of additional sites outside of the activation loop and ABCDE and PQR clusters may also be necessary for dissociation of DNA-PKCS from Ku and DNA ends (26). DNA-PKCS has two caspase cleavage sites (Asp2712-Asn2713 and Asp2982-Gly2983) (27;28). Cleavage of the protein, which separates the FAT/PIKK/FATC module from the DNA-binding HEAT repeats and results in loss of DNA-PK activity, was observed following treatment of cultured cells with apoptotic agents. This mechanism may favor DNA fragmentation and nuclear disassembly necessary for apoptosis, and confine DNA-PK activity to viable cells.
Several cryo-electron microscopy studies (10;29-33), and recent solution and crystal structures obtained by X-ray scattering (34) and X-ray crystallography (PDB: 3KGV) [Figure 4 & 5; (14)] identified three major structural domains in DNA-PKcs termed the head, arms, and palm [reviewed in (35)] (Figure 3). The head structure, comprised of the PIKK domain and the FAT and FATC domains, resides on top of the ring-shaped palm region, which is composed of two arms that form a circular structure around a central opening that presumably holds the double stranded DNA. The arms consist of the N-terminal two-thirds of the protein and contain ~66 helices arranged as HEAT repeats in a ring structure; when viewed from the side this structure forms a concave shape. The N terminus of the protein lies in the right arm, while the LRR is in the left arm as shown in Figure 3. A gap at the bottom of the ring is predicted to widen upon autophosphorylation of either the ABCDE or PQR clusters, releasing the DNA after ligation. The cryo-EM studies also indicated the existence of additional openings not observed in the crystal structure, possibly between the head and palm regions, which were postulated to bind single stranded DNA. The clover mutation affects the splice donor site within intron 29, and is predicted to cause skipping of exon 29. A transcript lacking exon 29 would encode the wild type sequence up to amino acid 1119, followed by four aberrant amino acids and a premature stop. This transcript would likely be subject to nonsense-mediated decay.
|
Expression/Localization | DNA-PKCS has been detected at mammalian telomeres in all cells (1;36). DNA-PK kinase activity appears to be cell cycle- and phosphorylation-dependent (e.g. it is reduced in S phase (compared to G1)) (37). DNA-PKCS has only been detected in higher eukaryotes (38). Interestingly, primate cells express 50 times more DNA-PK activity than rodent cells (23;39). Based on the interaction of DNA-PKCS with C1D, it has been suggested that DNA-PKCS may be sequestered in particular nuclear compartments (16).
|
Background | DNA DSBs can occur as a result of exposure to external factors including ionizing radiation (IR) (e.g. medical x-rays and radon gas decay in the soil) (40), radiomimetic drugs (e.g. the antibiotic, bleomycin) (41), toxins (e.g. asbestos, silica, and titanium dioxide) (42), and topoisomerase inhibitors (e.g. camptothecin, which traps enzyme-DNA intermediates and inhibits the re-ligation of DNA) (43). Cellular processes such as the generation of reactive oxygen species as a byproduct of oxidative metabolism, the collapse of DNA replication forks (upon recognition of single-stranded breaks by the replication machinery) (44), and, in the case of B and T lymphocytes, immune receptor gene arrangement, also cause DSBs (16;38;45;46). Repair of DSBs is required to prevent chromosomal abnormalities and chromosome loss, and thereby maintain genomic stability. If left unrepaired, cell cycle arrest typically occurs, leading to cell death (45;47;48). In addition, instances of cancer can occur after a tumor suppressor gene is inactivated or deleted by a DSB, or when an oncogene is activated or translocated (49;50).
DSB repair pathways
There are three DNA DSB repair pathways: single-strand annealing (SSA), homologous recombination (HR), and nonhomologous end-joining (NHEJ) (51). In SSA, which requires homologous sequences to flank the break site, the nonhomologous DNA ends are removed, leaving overhangs that are aligned and annealed (45). After annealing, DNA synthesis and ligation occur. SSA results in deletion of the region between homologous sequences. In contrast, HR is a relatively error-free mechanism for DSB repair that relies on the homologous region on the sister chromatid as a template for DNA synthesis (Figure 6A) (1;16). HR is limited to replicating cells (including early embryonic developmental stages) and can be observed only after DNA replication in the S and G2 phases (52) . Finally, NHEJ is a rapid repair mechanism that simply ligates broken DNA ends after minimal or no processing (Figure 6B). NHEJ occurs in all phases of the cell cycle and is thought to be the primary DNA repair pathway in mammalian cells (53;54). NHEJ is prone to generating mutations at the point of ligation, and may result in inversions or translocations if the joined strands were not originally contiguous. Nonhomologous end-joining (NHEJ)
Seven factors are necessary for NHEJ: Ku70, Ku80, DNA-PKCS, XRCC4 (X-ray repair cross-complementing 4), DNA ligase IV, Artemis, and XLF (XRCC4-like factor; also known as Cernunnos) (1;16;55). During a DSB repair, the break is recognized by the Ku heterodimer composed of Ku70 and Ku80, which encircles the DNA and cups the DNA termini into an accessible binding pocket (56). The Ku dimer can recognize blunt ends, hairpin ends, and 5’ or 3’ overhangs; two in vitro studies using purified oligonucleotides containing hairpin ends found that the Ku heterodimer (and DNA-PKCS) can bind hairpin ends but the kinase could not be activated (48;57). The Ku heterodimer recruits and activates DNA-PKCS, forming the Ku/DNA-PKCS complex known as DNA-PK. The Ku/DNA-PKCS interaction not only assists in localizing the complex to the DNA ends, it also protects DNA ends from nuclease digestion prior to re-ligation (45;58). Following recruitment of DNA-PKCS to the Ku-DNA complex, Ku translocates inward ~10 bp from the DNA ends, allowing DNA-PKCS to bind to the DNA termini (38). Two adjacent DNA-PKCS molecules interact across the DSB, holding the DNA ends in close proximity within a synaptic complex. Crystallographic studies have shown that the synaptic complex in NHEJ consists of two DNA ends, two Ku heterodimers, and two DNA-PKCS proteins (33;58). The formation of a synaptic complex is essential for the activation of DNA-PKCS (58). Nucleases (e.g. 5’: FEN1, EXO1, and Sep1 (59-61); 3’: MRE11 (62)) and polymerases (e.g. polβ, polε, and polδ) are often required to remove several nucleotides or to fill in gaps of several nucleotides, respectively, to facilitate the proper conformation for ligation (53). The colocalization of DNA polymerase X family members (e.g. terminal deoxynucleotidyl transferase (TdT), pol μ, pol λ, and polβ) with DNA-PKCS as well as the interactions of DNA pol X with both Ku and the DNA ligase IV-XRCC4 complex suggest that the DNA polymerase X family participates in the filling in of short gaps prior to re-ligation (1;23). To protect the DNA termini of a DSB from degradation or premature and incorrect ligation, DNA-PKCS is positioned as a “cap” on the DNA ends (18;63). Before re-ligation of the DNA ends and finalization of the DSB repair, the DNA-PKCS cap must be removed or altered. Autophosphorylation of DNA-PKCS results in release of the cap and accessibility of the termini to enzymes and ligases needed to complete the repair (e.g. Artemis, DNA polymerase X family members and the DNA ligase IV-XRCC4 dimer) (1;16;58). Artemis and DNA-PKCS form a complex with endonuclease activity that cleaves 5’ and 3’ overhangs during NHEJ, and opens hairpins generated by the RAG complex during V(D)J recombination [(64); reviewed in (16)]. In order to activate its nuclease activity, Artemis is phosphorylated by DNA-PKCS (65). The DNA ligase IV-XRCC4 dimer rejoins the DNA ends, with XRCC4 both interacting with and catalytically stimulating DNA ligase IV (16). XLF functions to stimulate the ability of XRCC4-DNA ligase IV to ligate in the presence of Mg2+ (66). In general, mutations in genes required for DNA damage repair can lead to radiosensitivity, immunodeficiency, cancer predisposition, and premature aging (38). More specifically, mutations in components of the DSB repair pathway cause a number of conditions including: LIG4 syndrome, radiosensitive severe combined immunodeficiency (RS-SCID), ataxia-telangiectasia (AT), AT-like disorder (ATLD), and Nijmegen breakage syndrome (NBS) (38). In LIG4 syndrome (OMIM #606593), there are developmental defects and growth retardation, immunodeficiency, and a predisposition to cancer due to mutations in Ligase IV (67-69). SCID (OMIM #602450) can be caused by a mutation in the gene encoding Artemis [DCLRE1C, (70)] as well as mutations in the genes encoding DNA-PKCS [PRKDC, (71)], and DNA ligase IV [LIG4, (72)]; mutations in recombination-activating gene 1 [RAG1] (Rag1, see maladaptive and huckle) and RAG2 lead to B cell-negative SCID (see OMIM #601457 for more details) (73). RS-SCID patients suffer from immunodeficiency and have a predisposition to cancer. In AT (OMIM #208900) and ATLD (OMIM #604391), patients have cerebellar ataxia and immunodeficiency as well as a predisposition to cancer due to mutations in the ATM and MRE11A genes, respectively (74;75). NBS (OMIM #251260) is due to a mutation in the gene encoding NBS1, another protein essential for DNA damage detection and response. NBS patients suffer from cancer predisposition as well as mental and growth retardation (76;77). Interestingly, cells deficient in DNA-PKCS or Artemis are sensitive to ionizing radiation, but do not have cellular growth defects (65). DNA-PK
DNA-PKCS was initially identified in HeLa cell extracts as a kinase activated by double stranded DNA and was subsequently designated a member of the PIKK family (78;79). DNA-PKCS activity is regulated by several different factors (Table 1). Table 1: Regulation of DNA-PK (adapted from (45))
Regulating protein
|
Function
|
Reference
|
C1D
|
Activates DNA-PK without the need for DNA termini; speculated to change chromatin structure to aid in DNA-PK localization to damage site
|
(80)
|
c-Abl
|
Phosphorylates DNA-PK which inhibits the interaction of DNA-PK with DNA in vitro
|
(81;82)
|
Caspase 3
|
Cleaves DNA-PKCS, resulting in inhibition of DNA-PKCS kinase activity during apoptosis
|
(27;28;83)
|
Chk1
|
Stimulates DNA-PK kinase activity towards p53; increases DNA-PK-dependent NHEJ in vitro
|
(84)
|
DNA-PKCS
|
Phosphorylates itself on multiple sites, which is necessary for NHEJ; autophosphorylation of the ABCDE cluster induces a conformational change that releases DNA-PKCS from the Ku-bound DNA and thereby decreases kinase activity
|
(16)
|
Heat shock transcription factor 1 (HSF1)
|
Stimulates DNA-PK kinase activity in vitro possibly by stabilizing DNA-PK/DNA interaction; may regulate DNA-PK after heat stress
|
(85-87)
|
High mobility group proteins 1 and 2 (HMG1 and 2)
|
Stimulate DNA-PK kinase activity in vitro possibly by promoting DNA-PK/DNA association
|
(88;89)
|
Ku70, Ku80
|
Stimulates DNA-PK activity; recruits DNA-PKCS to DNA
|
(90-92)
|
Ku80 auto-antigen-related protein (KARP-1)
|
Downregulates DNA-PK activity leading to IR sensitivity
|
(93)
|
Lyn tyrosine kinase
|
Binds to DNA-PKCS and prevents its association with Ku, resulting in inhibition of DNA-PKCS kinase activity
|
(9)
|
MDC1
|
Binds the DNA-PK complex; regulates DNA-PK autophosphorylation
|
(94)
|
Nuclear clusterin
|
Binds to Ku after IR-induced DNA damage and promotes cell cycle arrest and death
|
(95;96)
|
Poly (ADP-ribose) polymerase (PARP-1)
|
PARP-mediated ADP-ribosylation stimulates DNA-PK kinase activity
|
(97-99)
|
Protein kinase Cδ
|
Phosphorylates DNA-PKCS; inhibits DNA binding and DNA-PKCS-mediated phosphorylation of p53; may promote apoptosis
|
(100)
|
Protein phosphatase 1 and 2A
|
Dephosphorylates DNA-PKCS, reactivating the kinase activity of DNA-PK
|
(101)
|
Protein phosphatase 5
|
Dephosphorylates DNA-PKCS, regulating its sensitivity to radiation
|
(102)
|
XRCC4 and ligase IV
|
Promotes association of DNA-PKCS with Ku
|
(103)
|
In addition to its role in NHEJ (described above), DNA-PK functions in a variety of cellular processes that involve DNA regulation:
- Regulation of DSB repair pathway choice. Cells deficient in DNA-PKCS display elevated levels of HR likely due to a lack of NHEJ, supporting the idea that the NHEJ and HR pathways compete for substrates during DSB repair (104). However, chemical inhibition of DNA-PKCS resulted in inhibition of both NHEJ and HR (105). It has been proposed that chemical inhibition of DNA-PKCS generates a mutant protein that is unable to dissociate from the DNA ends, blocking both NHEJ and HR repair factors from accessing the DNA ends [(105); reviewed in (46)].
- V(D)J recombination. During lymphoid cell development, the process of V(D)J recombination generates a variable region exon to which is subsequently joined a constant region gene, together encoding either an immunoglobulin or T cell receptor chain (Figure 7). In V(D)J recombination, a trans-esterification reaction mediated by RAG1/RAG2 produces an excised DNA fragment with blunt signal ends and two covalently closed hairpins at each end of the coding regions that must be joined (1;45). Artemis is essential to opening hairpins for V(D)J recombination following phosphorylation by DNA-PKCS. To process the DNA ends and ligate coding regions, the cell uses the NHEJ pathway. In cells lacking DNA-PKCS, V(D)J recombination intermediates cannot be completely processed and ligated, leading to an accumulation of hairpin intermediates (106). This indicates that DNA-PKCS may be necessary for cleavage of hairpin intermediates as well as for the final end joining step (1).
- Telomere maintenance and elongation. Telomeres consist of short repetitive DNA sequences at the ends of eukaryotic chromosomes (107). They protect coding regions near chromosome ends from shortening during DNA replication and also distinguish chromosome ends from damaged DNA in the form of DSBs. The maintenance of functional telomeres is essential for genomic stability; telomere dysfunction can often lead to chromosomal rearrangements and cellular senescence (108;109). DNA-PKCS , together with Ku70 and Ku80, acts as a telomere cap that protects and distinguishes telomeric DNA from broken DNA ends. DNA-PKCS is localized to the end of chromosomes (45) and knockout of DNA-PKCS, Ku70, or Ku80 results in end-to-end fusions of chromosomes and chromosomal instability (110;111). These end-to-end fusions appear to result from the chromosome ends being recognized as DSB ends that need to be repaired (108), and not from loss of telomeric DNA. Mouse and human cells treated with a kinase inhibitor that is highly specific for DNA-PKCS had a high frequency of telomeric fusion (109). This study found that the kinase activity of DNA-PKCS is required to protect the ends of chromosomes and the processing of the leading-strand telomere after replication (109).
- Transcription control [reviewed in (2)]. In addition to the other roles of DNA-PKCS, it has also been implicated in transcription initiation. The DNA-PK complex can control transcription by inhibiting RNA-polymerase I (RNA-Pol I) via phosphorylation (112). DNA-PK also phosphorylates RNA-Pol II (90). In vitro, DNA-PKCS can phosphorylate several transcription factors (e.g. p53, Oct-1, c-Jun) (113). DNA-PK-mediated phosphorylation of heterogeneous nuclear ribonucleoprotein U (hnRNP-U) stabilizes specific mRNAs, promoting gene expression (114). indicating that this protein has a role in controlling transcription.
- Regulation of p53-mediated apoptosis [reviewed in (2)]. DNA-PKCS phosphorylates p53 at serine 15 and 37 (115), and possibly serine 46 (116). Cells that are DNA-PKCS defective are hypersensitive to p53-mediated apoptosis induced by IR (2;117;118). Under apoptotic conditions, p53 and DNA-PK form a protein complex (119) that may detect disruption of DNA replication (caused by nucleoside analog incorporation) and eventually signal for apoptosis (2).
|
Putative Mechanism |
There are several Prkdc-deficient model systems that have been characterized. For example, in DNA-PKCS-deficient cell lines, transfection of DNA-PKCS constructs with mutations within the kinase domain were unable to participate in NHEJ and V(D)J recombination (16). In addition, there are several animal models of SCID (e.g. mouse (120), Arabian horses (121), and Jack Russell terriers (122)) with mutations in Prkdc (123). Animals with SCID present with several features including: lymphopenia, hypogammaglobulinemia, premature aging (e.g. shortened telomeres), a large number of telomeric fusions, and impaired T and B cell-mediated functions (e.g. defective V(D)J recombination and reduced numbers of peripheral lymphocytes) (45;65;123). Mutations in Prkdc also lead to hypersensitivity to IR and radiomimetic chemicals (124). In horses with mutations in DNA-PKCS, decreased numbers of B and T lymphocytes were documented, although natural killer cell activity was normal (125). The decreased T and B cell functions were due to an inability of the lymphocytes to properly mature due to defective V(D)J recombination. 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 (126). Although a variety of mutations in Prkdc cause SCID in the aforementioned animal models, only one case of human SCID has been attributed to mutation of PRKDC (127). In that patient, two homozygous changes were verified: deletion of a glycine at 2113 and a missense mutation (L3062R) that was shown to be the disease-causing mutation . It was speculated that due to the differences in expression levels across species, a deficiency of DNA-PKCS in humans is incompatible with life (23). The clover mutation causes a similar phenotype to other Prkdc mutations: decreased T and B cell function likely due to a disruption in V(D)J recombination (Figure 8).
|
Primers |
Primers cannot be located by automatic search.
|
Genotyping | Clover genotyping is performed by amplifying the region containing the mutation using PCR followed by sequencing of the amplified region to detect the nucleotide change. The following primers were used for PCR amplification: Primers for PCR amplification
Clover (F): 5'- TGAGGCTGTTGGTCATGGACTTTCTA -3'
Clover (R): 5'- TGTGACACTCAGTCCTTATACTGCAAAATT -3' Primers for Sequencing
Clover_seq (F): 5'- CCAGCATGGTGGCTCTAAATC -3'
Clover_seq (R): 5'- CATCTTCAGCATTCATCAGATGGA -3' PCR program
1) 94° C 2:00
2) 94° C 0:30
3) 57° C 0:30
4) 72° C 1:00
5) repeat steps (2-4) 29x
6) 72° C 7:00
7) 4° C ∞ The following sequence of 1698 nucleotides (from Genbank genomic region: NC_000082 of the linear genomic sequence of Prkdc) is amplified: 63301 tgaggctg ttggtcatgg
63361 actttctatt tggatgtgat actccttttg ggtgtatgta tataatagat atgtgttgca
63421 gtaggcaatt aacctacact caaatcgccc tgacgtggca atgagagttc cttgctaccc
63481 acctcaatgt tctggattcc cgaatgaaag acacacacac agccctgtat ttaatatgcc
63541 ttaatcagct caatggttgg gccactacca aacctccacg tggctaacac ctcccttctg
63601 atactcctga attattactt actaaaatct atattccatc tttgttgccc tagacccagt
63661 ggggtctctg gctcccgatt gctgtgcatt cttttctgca cctctcatat ctggacctta
63721 ttgctttccc agcatggtgg ctctaaatca tgcttcctct cttggccccc ttgcccgtga
63781 atcttcgagt tccacctttg tctccctgcc agccattggc cgccaacaac tttattgact
63841 aatcagaacc aactgggggc aggaaccctc agcatctcac atatggattc tagaggaatt
63901 ttgggaaccc agttaacatc atacaagcat tagaccaaat tcaaattcaa attcacaaca
63961 tacatgcatg ttgtcatgcc ttctttcagt catgagtttg ggtttagttt aggatttgaa
64021 atgtaagtca tttattttaa tttcatattt tgagatgtag aaatactaaa tctatagtaa
64081 aattaccttg gtattcacaa aattcggtca gcagatttat aatattactg tgttaatgac
64141 aatctgattt gttttctatg atatatttct gtactggcca cacattcagg cacagttcag
64201 cagtgctgtg atgccatcga tcacctaaga cgcatcattg aaaagaagca tgtctcttta
64261 aacaaagcaa aaaagcgacg tttgccacag taggtgatac tgtttttctt cttttcttta
64321 acccaacaca aagctaattt ccatctgatg aatgctgaag atgaatatgt agaatataaa
64381 gaattatctt tgtaaggaag aaatgtgtag aagggtggta tgagttttct tttctttttt
64441 tttttcattt tttaaaatta gatactttat ttacagttca aattttatcc ccttccctcg
64501 tttccactcc aaaaaccctc ctatcccatc ccccctgccc cctgctcact aacccaccca
64561 ctctcgtttc cctgtcctgg cattcctcta cactggggca tcgagccttc acaaggccaa
64621 gggcctctcc tctcattgat gtcccacgag gccatcctct gctacatatg cagctgaagc
64681 cttgagtccc tccttgtgta ctctttggtt ggtggtttag ttcctgggag atcagggggt
64741 actggttggt tcatattgtt gttcctccta tggggctgca aaccccttca gctccttggg
64801 ttctttctct agctttattg gggagcagtc atttttctag caaaatttaa aacttttaag
64861 tttgaaagta catggtttca ggatgataat atttgattat ggtttggata ggtatccata
64921 ttgacagtaa gttttgaaca ttgtatttta cctgttacaa gttttgtaag tcattattta
64981 attagaatat ttatgaactg ttacacaata aattttgcag tataaggact gagtgtcaca PCR primer binding sites are underlined; Sequencing primer binding sites are highlighted; the mutated T is highlighted in red.
|
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Science Writers | Anne Murray |
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Authors | Carrie Arnold and Elaine Pirie |