DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair

Abstract

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 ...

Regulation and mechanisms of mammalian double-strand break repair

“…A recent report using an ISceI endonuclease-based cell system in human leukemic and lymphoblastoid cells demonstrated that expression of wild-type P53 in P53 null cells inhibits HRR as well as microhomology-directed NHEJ. Thus, P53 may restrain DNA exchange between imperfectly homologous sequences (Akyuz et al, 2002). More studies are needed to confirm these findings using chromosomal substrates instead of DSBs generated by a rare-cutting endonuclease and under conditions when other P53 effects are controlled.…”

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

“…We have previously shown that spontaneous recombination events, which are independent of any targeted cleavage but strictly associated with DNA replication, can be detected in cells carrying a stably integrated EGFP-based substrate (13,25) (Fig. 1A).…”

p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage

“…However, how these isoforms affect DNA damage repair is rarely studied. Many studies have demonstrated that full-length p53 inhibits DNA DSB repair [12][13][14]. A recent study using human cells has shown that, in response to γ-irradiation treatment, p53 pulses induce apoptosis at the early stage and postpone DNA DSB repair to the later stage [22].…”

“…The expression of p53 downstream genes triggers cell cycle arrest, DNA damage repair, apoptosis and/or senescence to ensure genome stability [7,8]. Intriguingly, p53 protein appears to promote only some DNA damage repair pathways, such as base excision repair, mismatch repair and nucleotide excision repair [9][10][11], but inhibit DNA DSB repair pathways, including the HR, NHEJ and SSA pathways [12][13][14]. It has been demonstrated that p53 exerts a direct effect on DNA DSB repair, as mutations in p53 that impair or even abolish its transcriptional activity and cell cycle regulatory capacity do not significantly affect its inhibition of HR [15][16][17].…”