Oncogene (2002) 21, 4788 – 4800 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Association of p53 and MSH2 with recombinative repair complexes during S phase Daniele Zink1, Christoph Mayr1, Christine Janz2 and Lisa Wiesmu¨ ller*,2 1Institut fu¨r Anthropologie und Humangenetik LMU Mu¨nchen, Goethestr. 31,80336 Mu¨nchen, Germany; 2Universita¨tsfrauenklinik und Poliklinik, Prittwitzstrasse 43, D-89075 Ulm, Germany Our previous recombination and biochemical analyses hower et al., 1992; De Wind et al., 1995; Reitmair et al., have led to the hypothesis that the tumor suppressor p53 1995). Synergistically increased cancer susceptibilities of monitors homologous recombination, a function which the double knockouts indicate complementary anti- was previously attributed to the mismatch repair protein carcinogenic activities for these multi-functional MSH2. Here, we show that a certain fraction of p53 is proteins (Cranston et al., 1997). In MSH27/7 mice concentrated within discrete nuclear foci of cells genomic instabilities are associated with mismatch synchronized in G1 phase, a pattern which becomes even repair deficiencies and a hyperrecombinative phenotype more pronounced in S phase, especially after g-ray (De Wind et al., 1995). Genomic instabilities in p537/7 treatment. p53 foci show some colocalization with mice are characterized by a high degree of aneuploidies, MSH2 within distinct foci during G1 phase, while dots gene amplification rates, and sister chromatid exchanges formed by BRCA1 display an independent localization (Livingstone et al., 1992; Ishizaki et al., 1994). pattern. In S phase nuclei, p53 foci almost completely A central role in maintaining the genome integrity was colocalize with MSH2 foci and associate with the attributed to p53, when it was understood that p53 halts recombination surveillance factor BRCA1 in irradiated the cell cycle or induces apoptosis in response to various S phase cells. These p53 and MSH2 foci also show stress signals (Lane, 1992; Levine, 1997). Transcriptional significant overlaps with foci of the recombination transactivation of target genes is required for the p53- enzymes Rad50 and Rad51, which for the first time mediated arrest in G1, in G2, as well as for many of the unveiled recombination-related functions of p53 in apoptotic responses. Most of the cancer-related p53- replicating cells. During S phase, p53 and MSH2 are mutants show defects in checkpoint control. Therefore, maximally active in binding to early recombination until recently, p53 was believed to exclusively avoid the intermediates, and coexist within the same nuclear manifestation of DNA damage by transcriptionally DNA-protein complexes. Our data suggest that p53 is signaling cell cycle arrest or apoptosis. However, further linked similarly to homologous recombination as MSH2 mechanisms might contribute to the genome stabilizing and provide further evidence for the new concept of a and tumor suppressor functions of p53, as indicated by dual role of p53 in the regulation of growth and repair. the absence of tumor formation after treatment of mice Oncogene (2002) 21, 4788 – 4800. doi:10.1038/sj.onc. with g-ray and a compound, which inactivates transcrip- 1205614 tional and apoptotic functions of p53 (Komarov et al., 1999). A direct participation of p53 in DNA repair Keywords: BRCA1-associated surveillance complex; processes is conceivable due to biochemical activities of Rad50; Rad51; replication-associated homologous re- p53 in DNA damage recognition, reannealing, exonu- combination; tumor suppressor cleolytic degradation, and due to interactions with replication and repair factors, such as the human strand transferase hRad51 and the breast cancer gene product BRCA1 (Albrechtsen et al., 1999). More specifically, an Introduction involvement of p53 in recombinative repair processes was indicated, because spontaneous homologous recom- MSH2 is a tumor suppressor linked to hereditary bination (HR) processes were deregulated in cells, in nonpolyposis colorectal cancer (HNPCC), alterations which p53 was inactivated by genetic alterations or by in the p53 gene are the most frequent mutations found in viral protein interactions (Wiesmu¨ ller et al., 1996; a variety of different tumors (Hollstein et al., 1994; Bertrand et al., 1997; Mekeel et al., 1997). The Modrich and Lahue, 1996). Consistently, deficits in identification of cancer-related separation of function either MSH2 or p53 cause the accelerated formation of mutations made it unlikely that the regulation of HR is tumors in the corresponding knockout mice (Done- merely an indirect effect of p53’s activities in transcrip- tion and growth control, and suggested that functions in recombinative repair contribute to tumor suppression *Correspondence: L Wiesmu¨ ller; (Saintigny et al., 1999; Dudenho¨ ffer et al., 1999; Willers E-mail: [email protected] et al., 2000). Mechanistic studies indicated that p53 Received 9 July 2001; revised 9 April 2002; accepted 26 April 2002 regulates HR events involving Rad51-dependent strand p53 and MSH2 at sites of recombination D Zink et al 4789 invasion rather than single-strand annealing processes and Lahue, 1996; Dudenho¨ ffer et al., 1998), we (SSA) and unveiled an effect of certain mismatched bases analysed the proteins after the introduction of DNA within the heteroduplex generated by strand exchange strand breaks by ionizing irradiation. Western blotting (Dudenho¨ ffer et al., 1998; Saintigny et al., 1999; Su¨ sse et and PhosphorImager evaluation with total lysates from al., 2000). This has led to the hypothesis that p53 exponentially growing CV1 monkey cells demonstrated monitors the fidelity of HR processes to avoid error- that the amount of wild-type p53 rose up to fivefold prone repair. within 1 h in response to irradiation at doses up to So far, fidelity control of recombination has been 12 Gy, whereas MSH2 levels increased less than ascribed to MSH2 and to its bacterial homologue MutS, twofold (Figure 1a). For comparison, Rad51 accumu- because these proteins inhibit DNA exchange processes lated 15-fold, and Rad50 less than twofold. Given that between divergent sequences beyond their roles in binding of recombination intermediates seems to be a sensing base mispairings during postreplicative central step for p53 and MSH2, to prevent HR mismatch repair (Modrich and Lahue, 1996). Like between diverged sequences (Alani et al., 1997; p53, MSH2 interacts with recombination junctions with Dudenho¨ ffer et al., 1998, 1999; Su¨ sse et al., 2000), we high affinity (Alani et al., 1997; Dudenho¨ ffer et al., performed gel shift analyses with nuclear extracts from 1998; Marsischky et al., 1999; Su¨ sse et al., 2000). In CV1 cells and branched three-stranded (3s) DNAs, further analogy to p53, the MSH2/MSH6 mismatch mimicking early recombination intermediates (Whitby repair complex functions in the signaling pathways of and Lloyd, 1995; Dudenho¨ ffer et al., 1998; see Figure the G2/M checkpoint and towards programmed cell 1b). Specificity for these artificial recombination death in response to some chemical carcinogens intermediates was ascertained by competition analyses, (Yamada et al., 1997; Durant et al., 1999; Fishel, 1999). which demonstrated that at least a 1000-fold molar BRCA1 represents another tumor suppressor, which excess of tRNA or of poly dI/dC were required to like p53 and MSH2 performs regulatory activities compete for binding, and by controls with dsDNA and during recombination, cell cycle progression, and the ssDNA (Figure 6) of the same sequence content. The induction of apoptosis (Venkitaraman, 1999). A role in participation of p53 from irradiated cells in complexes recombination was anticipated from the observation of with 3s DNA substrates was demonstrated by p53- associations with Rad51 and with Rad50-Mre11-p95 specific antibodies. Thus, the retardation of the major complexes after irradiation (Scully et al., 1997a; Zhong DNA-protein complex band (Figure 1b, lanes 1,5) was et al., 1999). More directly, it was recently demon- observed with the mAb DO1 (lane 2). Supershifting strated that BRCA1 directs double-strand break (DSB) with PAb1620 verified that p53 from CV1 cells binds 3s repair from nonhomologous end-joining (NHEJ) into DNA in its wild-type conformation (lane 3). Junction the nonmutagenic pathway of HR (Moynahan et al., DNA interactions by p53 were enhanced in the 1999). Rad50-Mre11-p95 complexes promote DSB presence of these antibodies, which is similar to the repair via both NHEJ and HR pathways (Haber, antibody-mediated activation of the cryptic sequence- 1998). Rad51, in eukaryotes, plays the central role in specific DNA binding activity (Hupp and Lane, 1994). the DNA-strand exchange processes of conservative PAb421, directed to the regulatory C-terminal end of HR pathways (Baumann and West, 1998). p53 had almost no effect (lane 4), which probably In our earlier studies (Dudenho¨ ffer et al., 1998, 1999; reflects the inaccessibility of the respective epitope by Su¨ sse et al., 2000), we have provided evidence for an posttranslational modifications (Hupp and Lane, involvement of p53 in recombination immediately 1994). After the introduction of DNA damage DNA- following Rad51-mediated strand transfer. This is p53-antibody complex formation was enhanced reminiscent of the proposed mechanism, by which twofold, as was quantified by phosphorimaging of the MSH2 abrogates Rad51-mediated strand exchange shifts and by normalizing the resulting values (n=3) upon encountering a mismatch