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 ...
PARP-1 is rapidly activated by DNA strand breaks, which finally leads to the modulation of multiple protein activities in DNA replication, DNA repair and checkpoint control. PARP-1 may be involved in homologous recombination, and poly(ADP-ribosyl)ation of p53 represents one possible mechanism that activates p53 as a recombination surveillance factor. Here, we examined the influence of PARP-1 on homology-directed double-strand break (DSB) repair by use of a fluorescence- and I-SceI- meganuclease-based assay with either episomal or chromosomally integrated DNA substrates. Surprisingly, the transient expression of both full-length PARP-1 and of a dominant negative mutant, retaining the DNA-binding but lacking the catalytic domain, down-regulated DSB repair in a dose-dependent manner. This effect was seen regardless of p53 status, however, with enhanced inhibition in the presence of wild-type p53. Taken together, our data reveal that PARP-1 overexpression counteracts DSB repair independently of its enzymatic activity and of poly(ADP-ribosyl)ation of p53 in particular, but synergizes with p53 in suppressing chromosomal rearrangements.
Heteroduplex joints represent intermediates of Rad51-dependent recombination processes, which are recognized by p53 with extremely high a nities, in a manner independent of the DNA sequence content. To determine the structural elements required for complex formation, we monitored DNA-binding by protection against restriction endonuclease cleavage. We show that wild-type (wt) p53 interacts with heteroduplex joints in the proximity of the¯exible junction. Association of p53 within this junction region was also observed with preformed Rad51-heteroduplex complexes, whereas SSB counteracted p53 binding. At a distance of 31 bp from the junction p53 established very few contacts with the heteroduplex, despite the presence of an A ± G mismatch. Consistently, p53-dependent exonucleolytic degradation decreased when we raised the distance between the junction and the heteroduplex terminus by 27 bp. Di erent from the cancer-related mutant p53(273H), which did not recognize the junction, tetramerization defective p53-1262 was protection competent but displayed reduced complex stability in gel shifts. Moreover, p53-1262 performed exonucleolytic activities towards ssDNA like wtp53, but reduced degradation of heteroduplex joints. These results suggest that during recombination wild-type p53, as a tetramer, stably binds to strand transfer regions, enabling the protein to exonucleolytically correct heteroduplex intermediates early after strand invasion.
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