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.In response to DNA damage the tumor suppressor p53 induces a transient cell cycle arrest by transcriptional transactivation of target genes or triggers apoptotic cell death by transcriptional transactivation-dependent and -independent pathways (25). The generation of mice nullizygous for p53 made clear that, in addition, p53 counteracts aneuploidies, allelic losses, sister chromatid exchanges, and gene amplifications (17,28). With respect to the underlying mechanism, a direct participation of p53 in DNA repair was proposed. This was due to biochemical observations that revealed activities of p53 in the recognition of DNA damage, in DNA reannealing, and in exonucleolytic DNA degradation (1). p53 also binds to a plethora of repair-related proteins. The meaning of most of these interactions is not yet clear. Thus, uncertainties exist, whether p53 participates in nucleotide excision repair by modulating TFIIH activities (12, 32, 48) or rather counteracts sister chromatic exchanges after UV irradiation (7, 17).More convincingly, several groups unanimously reported on 5-to Ͼ100-fold rate increases of spontaneous inter-and intrachromosomal homologous recombination (HR), when wildtype p53 (wtp53) was inactivated genetically or by interactions with viral ...

The tumor suppressor p53 transcriptionally transactivates cellular target genes that are implicated in growth control, apoptosis, and DNA repair. However, several studies involving p53 core domain mutants suggested that regulatory functions in recombinative repair do not require transcriptional transactivation and are separable from growth-regulation and apoptosis. Leu22 and Trp23 within the transactivation domain of human p53 play a critical role in binding basal components of the transcription machinery and, therefore, in the transactivation activity of p53. To further delineate whether p53 target genes are involved in recombination regulation, we ectopically expressed p53(22Q,23S) in p53-negative cell lines, which carry reporter systems for different homology-directed double-strand break (DSB) repair events. Like wild-type p53, p53(22Q,23S) efficiently downregulated homologous recombination on two chromosomally integrated substrates without affecting exchange on a substrate for the compound pathway of gene conversion and nonhomologous end joining. Only upon lowering the p53 protein to DNA substrate ratio by several orders of magnitude, we noticed a weak defect of a p53 transactivation domain mutant in DSB repair assays. In conclusion, molecular interactions of p53 within the N-terminal domain are not required to restrain DNA recombination, but might contribute to this genome stabilizing function.

Chromosomal translocations and retroviral integration events at breakpoint cluster regions (bcrs) have been associated with leukaemias. To directly compare the effect of different cis-regulatory sequences on recombination, we adapted our SV40 based model system to the analysis of correspondingly selected bcrs from the TAL1, LMO2, retinoic acid receptor alpha (RARalpha) and MLL genes. We show that a 399 bp fragment from the MLL bcr is sufficient to cause a 3-4-fold stimulation of spontaneously occurring DNA exchange and to respond to etoposide by up to 10-fold further elevated frequencies, i.e. to mimic the fragility of the 8.3 kb bcr during chemotherapy. To analyse the regulatory role of p53 in recombination involving leukaemia-related sequences, we stably expressed wtp53 and a transactivation negative mutant. Consistent with the proposed role of p53 as a suppressor of error-prone recombination, both p53 proteins down-regulated recombination with most of the sequences tested, even with the MLL bcr after etoposide treatment. Surprisingly, however, p53 stimulated recombination, in constructs carrying the RARalpha bcr fragment. This is the first study, which provides evidence for a stimulatory role of p53 in homologous recombination. Our data further indicate that inhibition of topoisomerase I can mimic the effects of p53 on stimulating recombination on the RARalpha bcr. Thus, these data also firstly describe a biological role of the biochemical interactions between p53 and topoisomerase I that may have implications for a gain-of-function phenotype of certain p53 mutants in genetic destabilization.