Mutagenesis vol. 19 no. 5 © UK Environmental Mutagen Society 2004; all rights reserved.
Involvement of human p53 in induced intrachromosomal recombination in Saccharomyces cerevisiae
Institute of Clinical Physiology CNR, Area della Ricerca CNR, Via Moruzzi 1, 56100 Pisa, Italy and 1Mutagenesis Laboratory, National Cancer Research Institute (IST), L. go R.Benzi 10, 16132 Genova, Italy
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Abstract |
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p53 tumor suppressor protein plays a key role in maintaining genomic integrity and regulating growth control following exposure to DNA-damaging agents. Moreover, it is likely to control genome stability by affecting homologous recombination. p53 can work as a transcription factor in Sacharomyces cerevisiae, therefore this organism represents a good genetic model in which to investigate the molecular mechanism and genetic control of DNA damage-induced recombination. We expressed wild-type human p53 and a mutated form lacking transcriptional activity in S.cerevisiae strain RS112, which carries a synthetic intrachromosomal recombination substrate, and the frequencies of spontaneous and DNA damage-induced homologous recombination were determined. While an increase in intrachromosomal recombination induced by both UV radiation and methylmethane sulphonate (MMS) was observed in yeast cells carrying the void plasmid, p53 expression significantly reduced recombination frequency. The mutated p53 significantly reduced UV-induced recombination but had no effect on MMS-induced recombination. Our results suggest that human p53 inhibits homologous recombination induced by UV and MMS by mechanisms involving stabilization and/or phosphorylation of the protein.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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p53 is the most prominent example of a tumour suppressor protein and its gene has frequently been found to be altered in a wide spectrum of human tumours (Bullock et al., 2000
). In response to various types of genotoxic stresses, p53 transactivates a number of genes by binding to specific DNA sequences, thereby arresting the cell cycle, repairing damaged DNA or inducing apoptosis, thus preventing proliferation of cells with damaged genes (el-Deiry, 1998). The cellular response to genotoxic agents is an increase in the level and activity of p53 (Levine, 1997). A variety of genotoxic stresses initiate signalling pathways that transiently stabilize the p53 protein, cause its accumulation in the nucleus and its activation as a transcription factor (Foord et al., 1991; Siegel et al., 1995). The DNA alkylating agents mitomycin C and methylmethane sulphonate (MMS), as well as UV radiation, decrease the level of MDM2 mRNA and protein, indicating that down-regulation of MDM2 may contribute to the stabilization of p53 in response to these agents (Khosravi et al., 1999; Inoue et al., 2001). The fine mechanism of p53 modification after exposure to DNA-damaging agents is still unclear; recent studies point to multiple post-translational modifications as mediators of these events in response to genotoxic stress through several potentially interacting, but distinct, pathways based on phosphorylation (Lakin and Jackson, 1999; Appella and Anderson, 2001). ATM phosphorylation on Ser15 activated by DNA damage is considered the most important modification stabilizing p53. Even though yeast does not have a protein homologous to p53, it contains Tel1 and Mec1, the homologues of ATM and ATR (Foiani et al., 2000), and a serine/threonine kinase YGR262c which has been demonstrated to be closely related to a human p53 protein kinase (Clemente et al., 1997; Abe et al., 2001).
In addition to controlling growth in response to DNA damage, p53 is likely to regulate genome stability by affecting homologous recombination (Mekeel et al., 1997; Akyuz et al., 2002; Saintigny and Lopez, 2002). Physical and genetic links have been established between p53 and several factors involved in recombination, such as Rad51 recombinase, BRCA1, BRCA2 and helicase BLM (Marmorstein et al., 1998; Wang et al., 2001). Further analysis showed that p53 specifically interacts with and preferentially degrades recombination intermediates in a RAD51-stimulated fashion (Lee et al., 1997; Dudenhoffer et al., 1998; Janz et al., 2002). p53 has been shown to recognize mismatches arising within recombination intermediates, suggesting that it may contribute to controlling the fidelity of homologous recombination (Dudenhoffer et al., 1998; Susse et al., 2000). As homologous recombination gives rise to genetic alteration and cancer, understanding the role of p53 in this mechanism offers great opportunities to develop more sensitive and accurate diagnostic/prognostic tools, as well as more efficient therapies for cancer.
DNA damage-induced checkpoints are highly conserved from yeast to mammals and although there is no p53 homologue in Saccharomyces cerevisiae, when human p53 is expressed in yeast it controls cell growth and can work as a transcription factor (Nigro et al., 1992; Scharer and Iggo, 1992). Because S.cerevisiae represents the ideal organism in which to investigate the mechanisms and factors involved in homologous recombination, we expressed wild-type or Cys242Tyr mutated human p53 in yeast and tested the influence on spontaneous and DNA damage-induced intrachromosomal recombination. To understand if p53 stabilization occurs in yeast, we checked the levels and phosphorylation state of both p53 forms after exposure to DNA-damaging agents. The Cys242Tyr mutated form has lost its DNA-binding activity and is frequently found in human tumours (Olivier et al., 2002).
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Materials and methods |
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Yeast strain and culture conditions
The diploid strain RS112 of S.cerevisiae (MATa/
ura3-52/ura3-52, leu2-3,112/leu2-
98 trp5-27/TRP arg4-3/ARG ade2-40/ade2-101 ilv-92/ILV HIS3::pRS6/his3-200 LYS/lys2-801) was provided by Robert Schiestl (UCLA, Los Angeles, CA) (Schiestl, 1989). Culture media were prepared according to standard procedures (Schiestl, 1989; Kaiser et al., 1994). Cells were routinely grown in YAPD liquid medium up to logarithmic phase (
5–8 x 108 cells/ml). Cultures were maintained at 4°C for 24 h before experiments and then transferred to fresh YAPD liquid medium supplemented with 10 mM KH2PO4 (Yurkow and McKenzie, 1993; Del Carratore et al., 2002). Cultures were incubated under semi-anaerobic conditions for 17 h at 30°C.
Plasmid construction
Plasmids pUS76 and pUS33 were constructed from pRS316 and pLS76. pRS316 is a 4887 bp plasmid harbouring the URA3 marker, the CEN6/ARS4 and the ß-lactamase (AmpR) gene; pLS76 is a 9098 bp plasmid containing the CEN6/ARS4, AmpR gene, the LEU2 selection marker and wild-type p53 cDNA under control of the constitutive alcohol dehydrogenase promoter (pADH). The 2974 bp PvuI fragment from pRS316 was ligated with the 4996 bp PvuI fragment derived from pLS76. Plasmid pUS76 (URA selection, pADH1p53) (7970 bp) was characterized by size and PvuI restriction pattern. The wild-type p53 status was determined by PCR amplifying the p53 cDNA open reading frame and testing it by gap repair assay (Inga et al., 1997). pUS76 transfected into a ura–strain confers URA prototrophy. pUS33 was constructed as described for pUS76 starting from pLS76 expressing a specific p53 mutant Cys242Tyr.
Yeast transformation
Strain RS112 was transformed with pUS76 and pUS33 using lithium acetate as previously described (Gietz and Schiestl, 1991). Transformants were selected on glucose SC-URA plates.
Determination of MMS and UV-induced intrachromosomal recombination
The diploid strain RS112 transformed with wild-type and mutated human p53 forms was used to study the effect of p53 on MMS- and UV-induced intrachromosomal recombination. Intrachromosomal recombination events occur between two truncated his3 alleles, his3-5', which is truncated at the 5' end, and his3-3', truncated at the 3' end (Figure 1). These alleles share 400 bp of homology and are separated by the LEU2 marker (Schiestl et al., 1988). Ninety-nine per cent of HIS3+ recombinants result by deletion of the LEU2 gene (Schiestl, 1989). To measure intrachromosomal recombination, yeast cells were grown in medium lacking leucine and plated onto SC-HIS after treatment. Cells were exposed to MMS as follows: 5 x 106cells in logarithmic growth phase were incubated in 5 ml of liquid YAPD overnight in the presence of 10, 20 or 50 µM MMS (Sigma, St Louis, MO) for 17 h at 28°C. Cells were then plated onto SC-HIS and SC dishes to score recombinants and survivors, respectively. UV exposure was carried out as follows: 5 x 107 cells in logarithmic growth phase in SC-URA-LEU selective medium were washed, suspended in 10 ml of water and exposed to 254 nm radiation using a HPW 125 Philips lamp at dose rates of 30, 90 or 120 J/m2. After treatment, cells were incubated for 90 s in liquid YAPD medium, washed twice, plated onto SC-HIS and SC dishes and incubated at 30°C for 3 days. Frequencies of recombination are reported as number of HIS3+ colonies per 104 survivors.
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3' single strand-specific exonuclease, to expose the flanking homologous sequences. Annealing of the complementary single strands occurs and removal of the non-homologous ends is followed by DNA synthesis and ligation. (III) One-sided invasion and single strand annealing are basically two versions of the same mechanism. One-sided invasion is initiated by a DSB in one of the duplicated homologous regions and 5'Data obtained with cells transformed with wild-type and mutated p53 were compared with those with no p53. Statistical significance was determined by Student's t-test.
Protein extract preparation and western blotting
Yeast cells expressing p53 forms or not were exposed to MMS or UV as described above. Cells were lysed by a modification of the method of Kimmerly (Kimmerly et al., 1988). About 108 cells were suspended in 0.5 ml of suspension buffer [50 mM KCl, 5 mM MgCl2, 0.1 M EDTA, 25 mM HEPES, 5 mM DTT, 0.3 M (NH4)2SO4, 10% glycerol, pH 7.4] plus 10 µl of protease inhibitor solution [4.4 mg phenylmethylsulphonyl fluoride, 62 mg pepstatin, 50 mg chemostatin and 725 ml DMSO in 1 ml H2O]. Cell lysis was performed by vortexing five times for 30 s with glass beads. Fifty milligrams of protein yeast extract were electrophoresed on a SDS–polyacrylamide gel and blotted onto nitrocellulose. After transfer, membranes were blocked for 1 h in 5% non-fat milk in Tris-buffered saline (20 mM Tris–HCl, 0.5 M NaCl, 0.15% Tween 20). p53 level was immunoassayed using an anti-human p53 polyclonal antibody developed by Dako (Copenhagen, Denmark) at 1:1000 dilution, p53 phosphorylation was examined using an anti-phosphoSer-15 antibody (Santa Cruz Biotechnology, Germany) at 1:1000 dilution. Anti-ß-actin (Santa Cruz Biotechnology) was used as a control at 1:1000 dilution. An anti-mouse secondary IgG antibody coupled to horseradish peroxidase was used at 1:5000 dilution. Antibody reactive bands were detected using an enhanced chemiluminescence Western Detection System (Amersham-Pharmacia, Germany).
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Results |
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In the present paper we wished to investigate the effect of wild-type and mutated human p53 on MMS- and UV-induced intrachromosomal recombination in yeast. To this end the RS112 strain of S.cerevisiae was transformed with pUS76 carrying wild-type p53 or pUS33 carrying the mutated Cys242 p53 form under ADH promoter control.
Induction of intrachromosomal recombination by UV and MMS in p53 expressing yeast
Cells transformed with wild-type p53 were exposed to 30, 90 or 120 J/m2 UV or treated with 20 or 50 µM MMS and intrachromosomal recombination was detected as reported in Materials and methods. UV increased recombination 37.3-fold at the highest dose, with 34.5 events induced in RS112 not expressing p53 as compared with none in the untreated control. In cells expressing wild-type p53, the highest UV dose induced a 14.1-fold increase in intrachromosomal recombination (17.1 events as compared with none in the control) (Table I).
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Results obtained with MMS showed that wild-type p53 had an effect similar to that observed in the UV experiments. Precisely, in cells transformed with the void plasmid, the highest MMS concentration increased recombination 22.7-fold (20.6 events as compared with untreated cells). In cells expressing wild-type p53, the highest MMS concentration increased intrachromosomal recombination 8.6 times, with 9.9 induced events. As wild-type p53 reduces homologous recombination frequency induced by both UV and MMS we investigated whether the same effect was true for a cancer-related mutant p53. To this end we measured homologous recombination in the RS112 strain transformed with a mutated form of p53 carrying the Cys242Tyr substitution. This mutation is localized in one of the four ligands that coordinate the Zn atom and is considered a conformational mutation which reduces the DNA-binding and transactivational activity of p53 in mammals (Campomenosi et al., 2001
). In cells expressing mutated p53, the highest UV dose induced a 21.6-fold increase in intrachromosomal recombination (23.9 events as compared with none in the control) (Table I). On the other hand, the highest MMS concentration enhanced recombination 17.1 times and induced 18.7 events as compared with none in untreated cells (Table II). These results indicate that in yeast the expression of both wild-type and mutated p53 were able to significantly decrease UV DNA damage-induced intrachromosomal recombination, although the effect is more evident with the wild-type form. MMS induced intrachromosomal recombination was reduced only by the wild-type form. Neither the wild-type nor mutated p53 affected spontaneous intrachromosomal recombination; in fact, the frequencies with void plasmid and in p53-transformed cells were not significantly different (Tables I and II).
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Effect of UV and MMS on p53 level
To test the effect of DNA-damaging agents on the level of p53 expressed in yeast, RS112 cells were irradiated with UV or treated with MMS and total cell extracts were analyzed by western blotting using an anti-human p53 polyclonal antibody as reported in Materials and methods (Figure 2). A dose-dependent increase in the intensity of the band recognized by anti-p53 was observed after UV irradiation, in cells transformed with both wild-type (Figure 2a) and mutated p53 (Figure 2c); no protein was recognized in cells transformed with the void plasmid (Figure 2a and c, lanes 1). ß-actin was used to demonstrate that equivalent protein levels were loaded on the gels in all experiments. A dose-dependent increase in the band intensity was also observed after MMS treatment in extracts from cells transformed with wild-type p53 (Figure 2b), while no increase was detected in cells transformed with the mutant form with respect to untreated cells (Figure 2d). These results support the hypothesis that the Cys242 residue is critical for p53 stabilization in response to MMS damage. It is known that in mammalian cells one of the major controllers of p53 stabilization is phosphorylation of Ser15 by ATM. To evaluate whether UV or MMS treatment could also induce p53 phosphorylation in yeast, a specific anti-phosphoSer15 antibody was used. A western blot analysis of cellular extracts from cells expressing wild-type and mutated p53 and treated with UV or MMS is shown in Figure 2 (low lanes, all panels). p53 Ser15 is increasingly phosphorylated with increasing doses of UV or MMS, however, phosphorylation of the mutated form is induced by UV and not by MMS.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In mammalian cells the tumor suppressor gene p53 plays a crucial role in maintaining the integrity of the genome after exposure to DNA-damaging agents. Recent data suggest that p53 may have a direct role in DNA repair (Akyuz et al., 2002
; Saintigny and Lopez, 2002) and on homologous recombination that is stimulated when the replication fork stalls at DNA breaks or unrepaired lesions (Janz and Wiesmuller, 2002). In humans p53 down-regulates homologous recombination independently of its transcriptional transactivation function (Gottifredi et al., 2001; Sengupta et al., 2003; Yoon et al., 2004). To evaluate the effects of p53 on spontaneous and DNA damage-induced homologous recombination events in yeast, we expressed wild-type or cancer-related Cys242Tyr mutated human p53 in the RS112 strain of S.cerevisiae. We demonstrated for the first time that wild-type p53 expression significantly reduced MMS- and UV-induced intrachromosomal recombination in diploid RS112 cells by up to 50%; this is to be considered a strong decrease, even greater than that observed in p53–/– mice following X-ray exposure (Aubrecht et al., 1999; Lu et al., 2003). Moreover, previous results have shown that intrachromosomal recombination is not decreased by other heterologous proteins and the effect appears to be specific for p53 (Del Carratore et al., 2000).
In mammals a variety of genotoxic stresses initiate signalling pathways that transiently stabilize p53 protein by phosphorylation and cause it to accumulate in the cell. We tested this possibility in yeast by studying whether the level of p53 was affected by DNA-damaging agents and here we demonstrate that wild-type p53 is accumulated in S.cerevisiae after treatment with both UV and MMS. These data suggest that in yeast accumulation of p53 also directly correlates with a decrease in homologous recombination. To verify that the observed effect is specific for wild-type p53, we used a mutant form of p53 carrying the Cys242Tyr substitution. Cys242, located in loop 3, one of the four ligands that coordinates the Zn atom (Cho et al., 1994), has been shown to abolish DNA-binding activity of p53 (Campomenosi et al., 2001). In the present paper we have demonstrated that this mutated p53 form is still able to significantly reduce UV-induced recombination, even though to a lesser extent than the wild-type form, indicating that in yeast transactivation capability is not required for this function. In contrast, mutated p53 had no effect on MMS-induced homologous recombination. Interestingly, we observed that the reduction in UV-induced homologous recombination by mutated p53 correlated with stabilization of the protein, while no stabilization of mutated p53 and no effect on homologous recombination was observed after MMS treatment.
The mechanisms by which the p53 level increases in the nucleus of mammalian cells and the potential contributions of MDM2 to this process have been widely studied, even if they remain incompletely clarified (Maya et al., 2001; Blattner et al., 2002). The phosphorylation of N-terminal residues, including Ser15, is thought to prevent MDM2 binding and leads to accumulation of p53 in mammalian cells (Kubbutat et al., 1998; Lakin and Jackson, 1999; Unger et al., 1999; Inoue et al., 2001). Because yeast cells do not contain a MDM2 homologue, p53 stabilization occurs by an as yet unknown mechanism. Our results indicate that stabilization always correlates with Ser15 phosphorylation, so it is possible that Ser15 phosphorylation is necessary to slow down p53 turnover in yeast as in mammals.
p53 phosphorylation on Ser15 in mammals has been shown to be required not only for stabilization but also for its interaction with proteins present at the DNA damage site (Sengupta et al., 2003). In humans the phosphoSer15 form of p53 interacts with the homologous recombination proteins RAD51 and RAD54 and the anti-recombinogenic activity can be attributed to the direct binding of p53 to RAD51 (Linke et al., 2003). We found that both wild-type and mutated p53 stabilization as well as a decrease in UV-induced homologous recombination correlates with Ser15 phosphorylation. These findings suggest that in yeast the phosphorylated form of p53 might interact with the homologous protein RAD51 and so decrease UV-induced recombination. The mutated p53 form was neither stabilized nor phosphorylated after MMS-induced DNA damage. How can we explain this different stabilization/phosphorylation of the mutated p53 form in yeast after two different kinds of DNA damage? In humans two different kinases, Ataxia Telangectasia Mutated (ATM) and Ataxia Telangectasia Related (ATR) are activated to phosphorylate p53 Ser15 after genotoxic treatments (Nakada et al., 2003). In yeast Tel1 and Mec1, the homologous counterparts of ATM and ATR, might phosphorylate p53 after DNA damage (Foiani et al., 2000). It is known that in yeast different genotoxic stimuli induce different checkpoints, UV damage induces a G1 while MMS induces an S/G2 checkpoint (Foiani et al., 2000), so it is also possible that MMS damage activates a kinase(s) which recognizes wild-type but not mutated p53.
A further consideration arises from recent studies showing that UV- and MMS-induced homologous recombination occur by different genetic pathways. UV-induced homologous recombination is dependent on RAD52, which is the major partner of RAD51, while MMS-induced recombination is independent of RAD52 (Galli et al., 2003). We could speculate that both p53 forms are able to interact with RAD51 after UV treatment, while the mutated form is not able to interact with the proteins involved in MMS-induced homologous recombination.
In summary, we provide evidence for the first time that human p53 decreases DNA damage-induced homologous recombination in yeast, suggesting that it interacts with this highly conserved repair mechanism. Although p53 does not exist in yeast as such, a lot of papers report experiments using a yeast-based system to score functional properties of human p53. Our study strongly suggests that yeast is a very powerful genetic model to investigate how wild-type and mutant forms of p53 interact to modulate homologous recombination providing further knowledge about as yet unknown mechanisms.
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Acknowledgments |
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The authors wish to thank Robert Schiestl for providing the yeast strain. The study was supported in part by the Associazione Italiana per la Ricerca sul Cancro (to G.F.) and in part by EC contract no. 17225-2000-12F1EDISPIT.
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Notes |
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2 To whom correspondence should be addressed. Tel: +39 050 3152778; Email: rdc{at}ifc.cnr.it
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Received on November 13, 2003; accepted on May 24, 2004.
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