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. 2011;6(8):e23053.
doi: 10.1371/journal.pone.0023053. Epub 2011 Aug 12.

ATR-p53 restricts homologous recombination in response to replicative stress but does not limit DNA interstrand crosslink repair in lung cancer cells

Bianca M Sirbu  1 , Sarah J Lachmayer, Verena Wülfing, Lara M Marten, Katie E Clarkson, Linda W Lee, Liliana Gheorghiu, Lee Zou, Simon N Powell, Jochen Dahm-Daphi, Henning Willers
Affiliations

ATR-p53 restricts homologous recombination in response to replicative stress but does not limit DNA interstrand crosslink repair in lung cancer cells

Bianca M Sirbu et al. PLoS One. 2011.

Abstract

Homologous recombination (HR) is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB). However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Transactivation-impaired p53 restricts subnuclear RAD51 foci formation in response to replication stress.
(A) Representative images of subnuclear RAD51 foci formation in H1299 cells stably expressing p53QS or p53-null cells treated with 5 mM thymidine (TdR) for 24 hours. (B) Impact of p53 status (null versus QS) on RAD51 foci formation in H1299 cells treated with 5 mM TdR for 24 hours. Bars represent mean with standard error based on 3 independent repeats. (C) Impact of p53 status on RAD51 foci formation in H1299 cells treated with 1 mM hydroxyurea (HU) for 24 hours. Bars represent mean with standard error based on 5 independent repeats. (D) Impact of p53 status on RAD51 foci formation in H1299 cells 6 or 16 hours (h) after treatment with 2 Gy ionizing radiation (IR). Bars represent mean with standard error based on 2–5 independent repeats. All y-axes indicate percentage of treated cells with at least 10 RAD51 foci per nucleus after subtracting the percentage of untreated cells with background levels of RAD51 foci. P-values are based on Student's t-test (two-tailed).
Figure 2
Figure 2. p53 has a differential effect on I-SceI-induced HR in the chromosomal pDT219 recombination substrate.
(A) Plasmid substrate pDT219 carries a copy of the bacterial gpt gene inactivated by insertion of an I-SceI recognition site into the unique KpnI site of pSV2-gpt. HR events were induced in 10.1 mouse embryo fibroblasts carrying pDT219 by co-transfection of an I-SceI expression vector and a homologous donor. pΔ2 is characterized by 202 bp and 2333 bp of uninterrupted sequence homology shared with pDT217, while pKEB1 only contains 188 bp and 250 bp of homology flanking the break site. Following co-transfection of pKEB1 or pΔ2 together with an I-SceI expression vector, recombinants were scored in a colony formation assay. (B) I-SceI-induced HR frequencies obtained with chromosomally integrated pDT219 are plotted against p53 status, (−) indicating p53-null, (+) indicating the of transactivation-deficient p53-A135V mutant which is functionally analogous to p53QS. Bars represent the geometric mean with SEM of 3–6 independent experiments. The relative suppression of HR in the presence of p53-A135V compared to p53-null cells is indicated for each of the two donor plasmids. The relative suppression of HR was compared using the unpaired t-test (two-tailed).
Figure 3
Figure 3. Genetic analysis of p53's effect on HR in response to replicative stress reveals a role of the ATM/ATR phosphorylation site serine 15 (S15).
(A) Illustration of N-terminal p53 mutations introduced by site-directed mutagenesis. The mutant constructs were stable expressed from a common chromosomal integration site in H1299 cells (see Materials and Methods). (B) Cell cycle distributions for H1299 clones stably expressing a p53 N-terminal mutant. TdR, 5 mM thymidine for 24 hours. (C) Effect of p53 status on thymidine induced RAD51 foci formation, analogously to the experiments shown in Figure 1. p-value, result of t-test (two-tailed) comparing p53QS to p53-null cells.
Figure 4
Figure 4. Kinetics of RAD51 foci formation reveals early suppressive effect of p53 in response to replication stalling.
The time course of induced RAD51 foci in thymidine treated H1299 clones was measured analogously to the experiments shown in Figure 1.
Figure 5
Figure 5. Implicating ATR in the p53-mediated suppression of HR.
(A) H1299 clones were treated with thymidine (5 mM for 24 hours) with or without concurrent caffeine (5 µM) or KU55933 (20 µM) treatment. (B) Western blot illustrating siRNA mediated depletion of ATR in H1299 cells. sc, scrambled siRNA control. (C) Effect of p53QS status and ATR depletion on RAD51 foci induction, measured analogously to Figure 1.
Figure 6
Figure 6. HR suppressive function of p53 is bypassed in the cellular response to DSB.
(A) Staining for γ-H2AX as a marker of DSB formation, illustrating increase in DSB in both H1299 clones within 4 hours after release from thymidine (5 mM for 24 hours). (B) Time course of RAD51 foci induction, analogously to Figure 4, following removal of thymidine. To illustrate the similar increase in RAD51 foci induction irrespective of p53 status, the percentage of cells with foci was normalized to 0 at time 0 hours (h), i.e., at time of removal of thymidine. (C) Impact of p53 status on RAD51 foci induced 4 hours after treatment with mitomycin C (MMC) (0.5 µg/ml for 1 hour). Y-axis indicates percentage of cells with at least 10 induced RAD51 foci per nucleus. Similar results were seen after 24 hours (data not shown). (D) Impact of p53 status on γ-H2AX foci formation 24 hours after treatment with MMC. Y-axis indicates percentage of cells with at least 20 induced foci per nucleus. (E) Clonogenic survival of H1299 clones with varying p53 status. All data points are based on 2–3 independent repeat experiments.
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