Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53

We and others reported previously that the tumor suppressor p53 down-regula tes spontaneous homologous recombination in chromosomally integrating plasm id substrates, but how p53 affects homology-dependent repair of DNA double- strand breaks has not been established. Furthermore, it has been hypothesiz ed that p53 may suppress homologous recombination by direct interaction wit h recombination intermediates, but it is not known whether p53 directly act s on extrachromosomal plasmid substrates. In the present study, we asked wh ether p53 can suppress extrachromosomal spontaneous and double-strand break -induced homologous recombination. A plasmid shuttle assay was employed uti lizing episomally replicating substrates, which carried mutated tandem repe ats of a CAT reporter gene. Spontaneous homologous recombination and homolo gy-dependent repair of double-strand breaks induced by the I-SceI nuclease led to reconstitution of the reporter. Extrachromosomal homologous recombin ation was found to proceed independently of the p53 status of isogenic mous e fibroblast lines, contrasting the p53-mediated suppression of chromosomal recombination. The lack of p53 effect applied not only to the dominating s ingle-strand annealing pathway, which is Rad51-independent, but also to Rad 51-dependent gene conversion events. Comparison of homologous and non-homol ogous recombination frequencies revealed similar contributions to the repai r of I-SceI-induced breaks irrespective of p53 status. Our results are cons istent with a model in which the regulation of homologous recombination by p53 is restricted to the highly ordered chromosomal chromatin structure. Th ese data may serve as a cautionary note for future investigations using sol ely extrachromosomal model systems to address DNA repair in intact cells.