Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair.
Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70(-/-) cells and ku80(-/-) cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase β (Pol β). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80(-/-) mice had a shorter life span than dna-pkcs(-/-) mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER.
Department of Molecular Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Yong Jun
YJ
Choi
Department of Molecular Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Han
H
Li
Department of Molecular Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Mi Young
MY
Son
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
Xiao-Hong
XH
Wang
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
Jamie L
JL
Fornsaglio
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America ; Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States of America ; Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, United States of America.
Robert W
RW
Sobol
Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America.
Moonsook
M
Lee
Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America.
Jan
J
Vijg
Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
Sandra
S
Imholz
Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
Martijn E T
ME
Dollé
Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
Harry
H
van Steeg
Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands ; MGC Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus MC, Rotterdam, The Netherlands.
Erwin
E
Reiling
Department of Molecular Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Paul
P
Hasty
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics*; D001080:Apurinic Acid / Q000235:genetics; D059766:DNA End-Joining Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000037:antagonists & inhibitors; D051747:DNA-Activated Protein Kinase / Q000172:deficiency / Q000235:genetics; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics*; D004843:Epistasis, Genetic; D005260:Female; D017353:Gene Deletion; D007211:Indoles / Q000494:pharmacology; D000072200:Ku Autoantigen; D008136:Longevity; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics; D010641:Phenotype; D017354:Point Mutation; D021122:Protein Subunits / Q000172:deficiency / Q000235:genetics; D011836:Radiation Tolerance; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C503406:7-nitro-1H-indole-2-carboxylic acid; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D007211:Indoles; D009687:Nuclear Proteins; D021122:Protein Subunits; D016159:Tumor Suppressor Protein p53; D001080:Apurinic Acid; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase