Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53

Abstract

We and others reported previously that the tumor suppressor p53 down-regulates spontaneous homologous recombination in chromosomally integrating plasmid substrates, but how p53 affects homology-dependent repair of DNA double-strand breaks has not been established. Furthermore, it has been hypothesized that p53 may suppress homologous recombination by direct interaction with recombination intermediates, but it is not known whether p53 directly acts on extrachromosomal plasmid substrates. In the present study, we asked whether p53 can suppress extrachromosomal spontaneous and double-strand break-induced homologous recombination. A plasmid shuttle assay was employed utilizing episomally replicating substrates, which carried mutated tandem repeats of a CAT reporter gene. Spontaneous homologous recombination and homology-dependent repair of double-strand breaks induced by the I-SceI nuclease led to reconstitution of the reporter. Extrachromosomal homologous recombination was found to proceed independently of the p53 status of isogenic mouse fibroblast lines, contrasting the p53-mediated suppression of chromosomal recombination. The lack of p53 effect applied not only to the dominating single-strand annealing pathway, which is Rad51-independent, but also to Rad51-dependent gene conversion events. Comparison of homologous and non-homologous recombination frequencies revealed similar contributions to the repair of I-SceI-induced breaks irrespective of p53 status. Our results are consistent with a model in which the regulation of homologous recombination by p53 is restricted to the highly ordered chromosomal chromatin structure. These data may serve as a cautionary note for future investigations using solely extrachromosomal model systems to address DNA repair in intact cells.

p53 protects from replication-associated DNA double-strand breaks in mammalian cells

“…Here, we found that p53À/À MEFs have more cells with RAD51 foci (Figure 2a), in agreement with a general increase in HR previously reported in p53 mutated cells (Bertrand et al, 1997;Mekeel et al, 1997;Xia et al, 1997;Saintigny et al, 1999;Willers et al, 2000;Saintigny and Lopez, 2002). However, we found that the numbers of HU-induced RAD51 foci were similar in p53 þ / þ and p53À/À MEFs.…”

“…Thus, it is possible that p53 maintains stability of the genome through its interaction with RAD51. Support for this notion also comes from the observation that p53-deficient cells exhibit increased levels of HR and elevated levels of the RAD51 protein (Bertrand et al, 1997;Mekeel et al, 1997;Xia et al, 1997;Saintigny et al, 1999;Willers et al, 2000;Saintigny and Lopez, 2002). In addition, the lethality of RAD51 and RAD51L1 knockout mice can be suppressed by additional knockout of p53 (Lim and Hasty, 1996;Shu et al, 1999).…”

“…p53 interacts and colocalizes with RAD51 at stalled replication forks (Sturzbecher et al, 1996;Buchhop et al, 1997;Susse et al, 2000;Sengupta et al, 2003). Also, p53 has been shown to suppress HR induced at replication forks (Bertrand et al, 1997;Mekeel et al, 1997;Xia et al, 1997;Saintigny et al, 1999;Willers et al, 2000;Saintigny and Lopez, 2002). Thus, it is possible that p53 maintains stability of the genome through its interaction with RAD51.…”

Disruption of p53 by the Viral oncoprotein HPV16-E6 does not Deregulate Chromosomal Homologous Recombination in a Transcriptional Interference-Free Assay System

“…Similar results were obtained with extra-chromosomal assays. 32 Genetic disruption of p53 also did not lead to an increased rate of SCE in HCT-116 cells or in other human cell lines, 24,25 nor did it affect the frequency of meiotic crossover events in mice. 23 Together, these findings indicate that p53 has no direct role in suppressing HR mechanisms.…”

“…2,[27][28][29] The exact mechanism by which p53 discriminates between ectopic HR and allelic HR or SCE is unclear. However, the lack of p53's effect on allelic HR or SCE together with the demonstration that neither RAD51-dependent nor RAD51-independent HR involving extra-chromosomal DNA was affected by the p53 status of the cell 24,[30][31][32] indicate that p53 does not block DNA strand exchange reactions in cells, and that regulation of ectopic HR by p53 may be restricted to the highly-ordered chromatin structure. 32 The ability of p53 to alter chromatin structure and repress transcription, 33 together with the well established link between transcription repression and decreased efficiency of intra-chromosomal HR (reviewed in ref.…”