DNA substrate dependence of p53-mediated regulation of double-strand break repair

TY - JOUR

T1 - DNA substrate dependence of p53-mediated regulation of double-strand break repair

AU - Akyüz, Nuray

AU - Boehden, Gisa S

AU - Süsse, Silke

AU - Rimek, Andreas

AU - Preuss, Ute

AU - Scheidtmann, Karl-Heinz

AU - Wiesmüller, Lisa

PY - 2002/9

Y1 - 2002/9

N2 - 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.

AB - 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.

KW - B-Lymphocytes

KW - Cell Line

KW - Cell Transformation, Neoplastic

KW - Codon

KW - DNA

KW - DNA Damage

KW - DNA Repair

KW - Deoxyribonucleases, Type II Site-Specific

KW - Genes, Reporter

KW - Genes, p53

KW - Green Fluorescent Proteins

KW - Humans

KW - K562 Cells

KW - Luminescent Proteins

KW - Models, Genetic

KW - Plasmids

KW - Recombinant Fusion Proteins

KW - Recombination, Genetic

KW - Saccharomyces cerevisiae Proteins

KW - Substrate Specificity

KW - Transcriptional Activation

KW - Tumor Suppressor Protein p53

M3 - SCORING: Journal article

C2 - 12167722

VL - 22

SP - 6306

EP - 6317

JO - MOL CELL BIOL

JF - MOL CELL BIOL

SN - 0270-7306

IS - 17

ER -