Journal Article

Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53

Henning Willers, Ellen E. McCarthy, Petra Hubbe, Jochen Dahm-Daphi and Simon N. Powell

in Carcinogenesis

Volume 22, issue 11, pages 1757-1763
Published in print November 2001 | ISSN: 0143-3334
Published online November 2001 | e-ISSN: 1460-2180 | DOI:
Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53

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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.

Keywords: DSB, double-strand break; HR, homologous recombination; IR, ionizing radiation; MEFs, mouse embryo fibroblasts; NHEJ, nonhomologous end-joining; SSA, single-strand annealing.

Journal Article.  5442 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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