DNA substrate dependence of p53-mediated regulation of double-strand break repair.
DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.
Akyüz
Nuray
N
Universitätsfrauenklinik, D-89075 Ulm, Germany.
Boehden
Gisa S
GS
Süsse
Silke
S
Rimek
Andreas
A
Preuss
Ute
U
Scheidtmann
Karl-Heinz
KH
Wiesmüller
Lisa
L
D001402:B-Lymphocytes; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000502:physiology; D017930:Genes, Reporter; D016158:Genes, p53; D049452:Green Fluorescent Proteins; D006801:Humans; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D015533:Transcriptional Activation / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*
D003062:Codon; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific