Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53.
Nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) entails two principal mechanisms: modification of DNA ends prior to ligation (error-prone rejoining) or precise ligation without modification if the DNA ends are complementary (error-free repair). Error-prone rejoining is mutagenic, because it can lead to destruction of coding sequence or to chromosomal aberrations, and therefore must be tightly regulated. Previous studies on the role of the p53 tumor suppressor in the regulation of NHEJ have yielded conflicting results, but a rigorous analysis of NHEJ proficiency and fidelity in a purely chromosomal context has not been carried out. To this end, we created novel repair plasmid substrates that integrate into the genome. DSBs generated by the I-SceI endonuclease within these substrates were repaired by either error-prone rejoining or precise ligation. We found that the expression of wild-type p53 inhibited any repair-associated DNA sequence deletion, including a more than 250-fold inhibition of error-prone rejoining events compared to p53-null cells, while any promoting effect of p53 on precise ligation could not be directly evaluated. The role of p53 in NHEJ appeared to involve a direct transactivation-independent mechanism, possibly restricting DNA end-modification by blocking the annealing of single strands along flanking stretches of microhomology. The inhibition of error-prone rejoining by p53 did not apply to the rejoining of DSBs induced by ionizing radiation. In conclusion, our data suggest that p53 restricts the mutagenic effects of NHEJ without compromising repair proficiency or cell survival, thereby maintaining genomic stability.
Dahm-Daphi
Jochen
J
Laboratory of Experimental Radiation Oncology, Department of Radiotherapy and Radiation Oncology, University Hospital Eppendorf, University of Hamburg, 20246 Hamburg, Germany.
Hubbe
Petra
P
Horvath
Fruzsina
F
El-Awady
Raafat A
RA
Bouffard
Katie E
KE
Powell
Simon N
SN
Willers
Henning
H
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D051379:Mice; D008969:Molecular Sequence Data; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D016159:Tumor Suppressor Protein p53; D004247:DNA