Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint.
Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3'-hydroxyl and 5'-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.
Kühne
Christian
C
International Center for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Padriciano 99, I-34000 Trieste, Italy. kuehne@icgeb.org
Tjörnhammar
Marie-Louise
ML
Pongor
Sándor
S
Banks
Lawrence
L
Simoncsits
András
A
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D000072200:Ku Autoantigen; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012377:Rodentia; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C101467:XRCC4 protein, human; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen