Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control Henning Willers 1,2 , Ellen E McCarthy 1 , Biao Wu 1 , Hannah Wunsch 1 , Wei Tang 1,3 , Danielle G Taghian 1 , Fen Xia 1 and Simon N Powell* ,1 1 Laboratory of Molecular and Cellular Radiation Biology (MCRB), Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, MA 02129, USA; 2 Department of Radiation Oncology, University Hospital Eppendorf, 20246 Hamburg, Germany; 3 Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA The tumor suppressor p53 is considered as the guardian of the genome which is activated following genotoxic stress. In many cell types, p53 mediates G1 cell cycle arrest as the predominant cellular response. Inactivation of wild-type p53 leads to loss of G1/S checkpoint control and to genomic instability, including increased sponta- neous homologous recombination (HR). To determine whether regulation of the G1/S checkpoint is required for suppression of HR, we assessed recombination events using a plasmid substrate that stably integrated into the genome of p53-null mouse fibroblasts. Exogenous expression of a temperature-sensitive p53 protein (Ala135 to Val), which had lost trans-activation function and could not regulate G1/S transition when in mutant conformation, reduced HR rates to the same extent as wild-type p53. Furthermore, a p53 construct with an alternatively-spliced carboxy terminus also retained this ability in the absence of both activities, G1/S control and non-sequence specific DNA binding as mediated by the carboxy terminus. Our data dissociate regulation of HR by p53 from its role as a cell cycle checkpoint protein. The results support a model which extends p53’s role as a guardian of the genome to include transactivation- independent regulatory functions in DNA repair, replica- tion and recombination. Oncogene (2000) 19, 632 – 639. Keywords: p53; homologous recombination; G1/S cell cycle checkpoint; genomic stability Introduction The tumor suppressor gene p53 is viewed as the guardian of the genome (Lane, 1992) which regulates the cellular response to various stress signals, most notably exogenous DNA damage. In unstressed cells, p53 appears to be present at low levels and is assumed to exist in a latent form that requires modification to become active (Giaccia and Kastan, 1998). Activated p53 acts as a transcription factor that up-regulates several downstream genes. In many cell types, the dominant cellular response pathway is the G1 cell cycle arrest which is mediated by up-regulation of p21 (Kastan et al., 1992; El-Deiry et al., 1993) and which is assumed to allow for adequate repair of damaged DNA before entering S phase (Lane, 1992; Sherr, 1996). In certain cell types and situations, p53 can alternatively trigger apoptosis (Ko and Prives, 1996; Sherr, 1996). p53 has also been reported to be involved in control of the G2/M checkpoint and a variety of other aspects of cell proliferation and DNA metabo- lism (Ko and Prives, 1996; Levine, 1997; Janus et al., 1999a). Suppression of spontaneous homologous recombination (HR) has recently been established as a new endpoint of wild-type p53 function (Meyn et al., 1994; Xia et al., 1994; Wiesmu¨ller et al., 1996; Bertrand et al., 1997; Mekeel et al., 1997; Duddenho¨er et al., 1998). In a DNA repair pathway, recombinational processes may act to maintain genetic stability, but if deregulated or increased, genomic instability and malignant transformation can result. Importantly, increased HR could be a mechanism involved in loss of heterozygosity which is frequently observed in tumor development and progression (Cavenee et al., 1983; Koufos et al., 1984; Eyfjo¨rd et al., 1995). Thus, suppression of spontaneous HR is a means by which p53 can maintain genome stability. The underlying mechanisms of how p53 is involved in regulation of HR in-vivo have not yet been identified. Control of the G1/S cell cycle checkpoint could be hypothesized as the main means by which p53 may indirectly suppress HR events. An increase in HR frequency following inactivation of wild-type p53 might accordingly be due to replication on DNA lesions leading to generation of recombinogenic structures or due to uncontrolled entry of cells already containing recombination intermediates into S phase, which may result in an increase of genetic exchanges. In the current study, we therefore addressed the following questions: (i) Is suppression of HR related to p53-dependent cell cycle checkpoint control? (ii) If p53 has a more direct function in regulation of HR, does the basic C-terminal domain constitute the responsible regulatory domain, considering that its biochemical properties appear closely related to recombination processes, i.e., binding to single- and double-stranded DNA, recognition of DNA mis- matches, reannealing and strand transfer (Ko and Prives, 1996; Levine, 1997)? Analogous to our previous work (Mekeel et al., 1997), we assessed HR events occurring in a plasmid substrate which had been transfected into mouse fibroblasts. We found in-vivo evidence for a novel function of p53 in regulation of spontaneous HR that is distinct from its role as a cell *Correspondence: SN Powell, Department of Radiation Oncology, Cox 3, Massachusetts General Hospital Cancer Center, 100 Blossom Street, Boston, Massachusetts, MA 02114, USA Received 26 March 1999; revised 5 August 1999; accepted 9 August 1999 Oncogene (2000) 19, 632 – 639 ª 2000 Macmillan Publishers Ltd All rights reserved 0950 – 9232/00 $15.00 www.nature.com/onc