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ã Oncogene (1999) 18, 7706 ± 7717 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Maintenance of genomic integrity by p53: complementary roles for activated and non-activated p53 Nils Albrechtsen1, Irene Dornreiter1, Frank Grosse2, Ella Kim1, Lisa WiesmuÈller1 and Wolfgang Deppert*,1 1 Heinrich-Pette-Institut fuÈr Experimentelle Virologie und Immunologie an der UniversitaÈt Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany; 2Institut fuÈr Molekulare Biotechnologie, *Abt. Biochemie, Beutenbergstr. 11, D-07745 Jena, Germany In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53, through its various biochemical activities and via its ability to interact with components of the repair and recombination machinery, actively participates in various processes of DNA repair and DNA recombination. An important aspect in evaluating p53 functions arises from the ®nding that the p53 core domain harbors two mutually exclusive biochemical activities, sequencespeci®c DNA binding, required for its transactivation function, and 3'-45' exonuclease activity, possibly involved in various aspects of DNA repair. As modi®cations of p53 that lead to activation of its sequencespeci®c DNA-binding activity result in inactivation of its 3'-4 5' exonuclease activity, we propose that p53 exerts its functions as a `guardian of the genome' at various levels: in its non-induced state, p53 should not be regarded as a non-functional protein, but might be actively involved in prevention and repair of endogenous DNA damage, for example via its exonuclease activity. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. The dual role model for p53 in maintaining genomic integrity signi®cantly enhances p53's possibilities as a guardian of the genome. Keywords: p53; DNA repair; DNA damage; DNA recombination; DNA replication; sequence-speci®c DNA binding Introduction Twenty years after its discovery, the tumor suppressor p53 has become one of the most prominent molecules in the area of cancer research. Initially classi®ed as an oncogene (reviewed in Deppert, 1994) p53 meanwhile is regarded as a `master tumor suppressor' which ensures the integrity of the cells' genome by protecting it from adverse eects of DNA damage. Such damage triggers the activation and accumulation of p53 (Fritsche et al., 1993; Kastan et al., 1991; Nelson and Kastan, 1994). The best studied function of an activated p53 is that of a transcription factor, mediating expression of a variety of genes that induce a growth arrest or apoptosis. As a *Correspondence: W Deppert result, DNA damage will be repaired during a growth arrest, or the damaged cells will be eliminated, thereby preventing ®xation of DNA damage as mutations. This function of p53 led to the now famous coining of p53 as the `guardian of the genome' by Lane (1992). Although the main features of p53's role in maintaining the integrity of the genome seem to be outlined, there is still a lot to be learned as to how p53 exactly acts to prevent the accumulation of mutational events within a cell. Despite some uncertainties regarding the various mechanisms of its induction, elimination of damaged cells by p53-induced apoptosis is an easily conceived mechanism for achieving this goal. Much less is known about the role of p53 in DNA repair. So far, the main emphasis has been given to pathways leading to the activation of p53 by signals emanating from damaged DNA, with p53 integrating these signals and triggering a cascade of responses leading to either growth arrest or apoptosis. These mechanisms have been summarized in detail in several recent reviews (for example see Cox and Lane, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine, 1997; Bates and Vousden, 1999), thus this topic will be addressed here only brie¯y. The next steps in this cascade, namely the activation of the repair pathways themselves, and the role p53 plays in their activation are far less clear. Furthermore, it is still not known, whether and how p53 directly participates in DNA repair processes, despite some evidence pointing to this possibility. Last but not least, a possible role of p53 in the control of genomic integrity in its non-induced state, i.e., in the absence of signals indicating DNA damage or other potentially harmful conditions, so far has not yet been considered at all. In contrast, the general assumption is that a non-activated p53 is a non-functional one (Hupp and Lane, 1994; Vogelstein and Kinzler, 1992). In this review we will focus on p53-induced pathways that lead to or are part of DNA repair processes, with an emphasis on the known biochemical activities of p53, which might play a direct role in repair processes. Recent evidence from our laboratory showed that at least one of these activities, the 3'-45' exonuclease activity, is regulated in a manner opposite to that of sequence-speci®c DNA binding (Janus et al., 1999). Based on this ®nding we propose a model according to which p53 in its non-induced state, i.e. in the absence of conditions which lead to its activation, exerts basic functions in maintaining genomic integrity. Upon various cellular stress conditions, such as hypoxia, nutrient depletion, or DNA damage, the well known functions of p53 in integrating the respective signals become activated, triggering the Dual role model for p53 function N Albrechtsen et al cascade of p53 responses which lead to growth arrest, DNA repair, or apoptosis (Hall et al., 1996). Activation of p53 upon DNA damage Cell cycle check point control and apoptosis Eukaryotic cells have developed a network of highly conserved surveillance mechanisms (checkpoints) which ensure that damaged chromosomes are repaired before they are replicated or segregated. These mechanisms are essential for maintaining genomic integrity and cell viability. The tumor suppressor p53 is one of the critical mediators of cellular responses to various types of genotoxic stress (reviewed e.g. in Hall et al., 1996), acting at dierent levels of control during the cell cycle. In response to DNA damage, like ionizing irradiation (IR) (Kastan et al., 1991), ultraviolet (UV) irradiation (Maltzman and Czyzyk, 1984; Nelson and Kastan, 1994), hypoxia (Graeber et al., 1994), and ribonucleoside triphosphate depletion (Linke et al., 1996), p53 becomes activated as a transcription factor via speci®c post-translational modi®cations. The p53 protein is phosphorylated by a wide variety of protein kinases: in its transactivation domain by casein kinase (Ck) I, DNA-PK, ATM, JNK, and MAP kinases, and at its C terminus by cyclin dependent kinases (Cdks), PKC, and Ck II (see Martinez et al., 1997; Meek, 1994 and references herein). The dierent protein kinases can modify the sequence-speci®c DNA binding activity of p53 and may thereby modulate the relative eciency of activation of dierent p53 target genes (Hecker et al., 1996; Hupp and Lane, 1994; Hupp et al., 1992; Takenaka et al., 1995; Wang and Prives, 1995). Phosphorylation by DNA-PK within the transactivation domain of the protein (Shieh et al., 1997) also abrogates its interaction with MDM2, a key player in negative regulation of the transcriptional activity and the level of p53 (reviewed in Lane and Hall, 1997). DNA-PK phosphorylation on serine 15 and possibly serine 37 within (human) p53 correlates with the enhanced transcription of downstream p53 target genes. The ability of p53 to transactivate downstream target genes results in cell cycle arrest at speci®c points in the cell cycle. As a consequence, the cell cycle stops either before DNA replication in G1, or before mitosis in G2, which leads to a severe decrease in the amount of S phase cells. G1 arrest The p53-dependent G1 arrest results mainly from transactivation of the waf1 gene that codes for the small kinase inhibitor p21Waf1. p21Waf1 interferes with cell-cycle progression and prevents S phase entry by blocking the activity of Cdks (Dulic et al., 1994; ElDeiry et al., 1993; Harper et al., 1993; Xiong et al., 1993). Irradiated G1 phase cells accumulate high levels of Cdk2/cyclin E complexes which are inactivated by the association with p21Waf1. Inhibition of G1 phasespeci®c kinase activity maintains a hypophosphorylated retinoblastoma susceptibility gene product pRb, which represses E2F-speci®c transcription of genes that are required for entry into S phase, and thereby inhibit cell cycle progression. p53 may also be able to induce a G1-arrest by nontranscriptional mechanisms. Recently, it has been observed that p53 binds cyclin H, which is part of the Cdk7/cyclinH/Mat1 Cdk activating kinase complex (CAK). CAK plays a crucial role in activating cell cycle progression by phosphorylation and activation of Cdk2, and, as part of the TFHII complex, by controlling transcriptional activity of RNA polymerase II. In the TFHII complex, CAK phosphorylates the carboxy-terminal repeat domain (CTD) of RNA polymerase II, which is required for the transcriptional elongation by RNA polymerase II. Binding of p53 to CAK results in strong reduction of CAK activity both towards cdk2 and towards the CTD of RNA polymerase II, which could lead to either growth arrest or apoptosis (Ko et al., 1997; Schneider et al., 1998). S phase arrest In another checkpoint response pathway which operates in S phase and slows down the rate of DNA replication, p21Waf1 binds to the proliferatingcell nuclear antigen (PCNA) and blocks its activity, interfering with cell-cycle progression by blocking the elongation step in DNA replication (Waga et al., 1994). Stillman and coworkers suggested a dual role for PCNA in DNA replication and DNA repair which allows p21Waf1 to arrest DNA replication while permitting active DNA repair (Li et al., 1994a). In DNA replication, PCNA together with replication factor C (RFC) recognizes a primer-template junction and promotes loading of polymerase d (pol d). The PCNA-RFC-pol d complex also enhances the processivity of pol d during the elongation step of DNA replication (Melendy and Stillman, 1991). The direct binding of p21Waf1 to PCNA causes a rapid dissociation of the PCNA-RFC-pol d complex from the replication fork, stalling replicative DNA synthesis. In repair DNA synthesis, PCNA helps the polymerases d or e to localize the junction of incised DNA, but is not needed during DNA synthesis (Li et al., 1994a). Thereby, repair DNA synthesis could proceed also in the presence of an upregulated p21Waf1. G2 arrest The third and the last line of defense against induced chromatid damage occurs in G2. A possible role for p53 in the G2/M checkpoint was suggested by Li et al. (1994b) who reported that p21Waf1 can associate with cyclin A and cyclin B complexes during the later phases of the cell cycle, suggesting a functional interaction with the respective associated kinases. Furthermore, a bimodal periodicity for waf1 mRNA levels in human ®broblasts with peaks in G1 and G2/M was observed, proposing that p21Waf1 may play a role at the onset of mitosis. p21Waf1 is absent from the nucleus during S phase and transiently reenters the nucleus during late G2 phase. In late G2 half of the Cdk2/cyclin A is complexed with p21Waf1. p21Waf1 may thereby either directly inhibit the active kinase or prevent its activation by the Cdk-activating kinase CAK (Poon et al., 1996). Another possibility is that complex formation with p21Waf1 may block interaction of substrates with Cdk2/cylin A (Adams et al., 1996). In Xenopus egg extracts Cdk2 serves as a positive regulator for the activation of Cdk1/cyclin B complexes and prevents entry into mitosis when complexed to p21Waf1 (Guadagno and Newport, 1996). Nuclear accumulation of p21Waf1 and the resulting inactivation of Cdk-cyclin complexes at the onset of 7707 Dual role model for p53 function N Albrechtsen et al 7708 mitosis might be part of a p21Waf1 dependent mitotic attenuation mechanism (Dulic et al., 1998). However, p21Waf1 accumulation resulting from p53 activation seems to be mainly required for the DNA damageinduced G1 arrest, as p21Waf1 clearly is not essential for the immediate G2 checkpoint response (Brugarolas et al., 1995; Levedakou et al., 1995; Paules et al., 1995). Nevertheless, several studies have suggested that the G2/M arrest following DNA damage is also p53dependent (Agarwal et al., 1995; Aloni-Grinstein et al., 1995; Goi et al., 1997; Schwartz et al., 1997; Stewart et al., 1995). Vogelstein and colleagues used a wild-type p53 expressing human colorectal cancer cell line to analyse gene expression following g-irradiation. The cells arrested mostly in G2, and the block was accompanied by changes in gene expression. Quantitative analysis of gene expression patterns revealed a strong induction of the 14.3.3s gene. The induction of 14.3.3s is mediated by a p53-responsive element in the 14.3.3s gene (Hermeking et al., 1997). The 14.3.3-family proteins are found in a wide variety of mammalian tissues and in other eukaryotic organisms including plants and yeast (Aitken et al., 1992), and diverse biochemical properties have been ascribed to them. Mammalian cells contain a minimum of seven 14.3.3 isoforms (Aitken et al., 1995; Wang and Shakes, 1996). Studies in yeast indicate that 14.3.3 proteins have a role in determining the timing of mitosis (Ford et al., 1994). The induction of 14.3.3s by p53 might be part of p53's control of G2/M transition. The link between DNA damage control and cell cycle checkpoint control at G2/M could be further substantiated by the following observations. In irradiated cells the protein kinase Chk1, which is required for this DNA damage checkpoint (Sanchez et al., 1997), undergoes a Rad3dependent phosphorylation (Rad3 is a kinase related to the ATM protein) (Ford et al., 1994). The ultimate target of this checkpoint signal is thought to be Cdk1, the cyclin-dependent kinase that induces mitosis (Pines, 1991), as Chk1 directly phopshorylates Cdc25C, a phosphatase that regulates Cdk1 activity (Furnari et al., 1997; Peng et al., 1997; Sanchez et al., 1997). This phosphorylation event promotes the binding of 14.3.3 to Cdc25C and thereby its sequestration. In this state Cdc25C cannot dephosphorylate and activate Cdk1. Thus cell cycle progression after g-irradiation (and possibly other DNA damage inducing events) is blocked by independent p53-mediated eector pathways: Entry into S phase is inhibited by p21Waf1, whereas 14.3.3s prevents cells that have completed S phase from entering mitosis. Therefore the p53dependent induction of 14.3.3s connects DNA damage with G2/M progression driven by Cdk1 in the same way as the p53-dependent induction of p21Waf1 connects DNA damage with the Cdks required for G1/ S progression. Apoptosis In response to DNA damage, p53 can also induce apoptosis. Induction of apoptosis by p53 is complex and not yet understood in detail. Transcriptional activation of a variety of dierent genes involved in mediating an apoptotic response (e.g. bax, Fas/Apo, Killer/DR5, and redox regulator genes [PIGs]) again seems to be the major mechanism of p53-mediated apoptosis, but p53 is able to induce apoptosis also in a transcription-independent mode (Caelles et al., 1994; Haupt et al., 1995; reviewed in Bates and Vousden, 1999). The interaction of p53 with CAK already described above could be one such mechanism, but other interactions, mediated by the N-terminal prolinerich domain of p53 which serves as a docking place for proteins containing SH3-domains, have also been implicated. Even less understood is what determines the `decision' to induce either a growth arrest or apoptosis after p53 activation. The outcome de®nitely is cell type speci®c, and is also regulated by the severity of DNA damage, with heavier damage promoting apoptosis over growth arrest. Induction of growth arrest rather than apoptosis on the other hand is favored by the presence of survival factors. In this respect, it is interesting that IGF-BP3 is a target for transcriptional activation by p53. Overexpression of IGF-BP3 can inhibit the mitogenic activity of IGF receptor signaling, while in other systems it can inhibit the survival signals provided by IGF, thereby amplifying apoptosis. Thus the extracellular environment and intracellular signals strongly modulate the p53 response (reviewed in Bates and Vousden, 1999). Selectivity of transcriptional regulation by p53 As the outcome of the cellular response upon activation of p53 to a great extent is dependent on transactivation of the appropriate target genes, the question arises how selectivity of transactivation is secured. So far, approximately 20 more or less con®rmed target genes of p53 have been described, which will induce quite dierently and even opposing or mutually exclusive cellular responses. Thus it is necessary that p53 is able to discriminate between those genes, activating transcription from only those target genes which mediate the appropriate response in a given cellular environment and physiological situation. Several ways to achieve this goal can be envisioned. It has been proposed that dierent posttranslational modi®cations of the p53 protein will lead to the preferential activation of certain target genes (reviewed in Jayaraman and Prives, 1999). Similarly, the association of p53 with dierent co-activator molecules will confer speci®city and selectivity to p53-mediated transactivation. For example, association of p33ING with p53 seems to be required for the ecient transactivation of the waf1-promoter by p53 (Garkavtsev et al., 1998). In addition to these mechanisms our laboratory recently suggested a novel way to ensure speci®city and selectivity of transactivation by p53. The cognate DNA sequences in p53-responsive promoters are characterized by signi®cant sequence-heterogeneity, as the consensus sequence, consisting of two 5'-PuPuPuC(A/T)(TA)GPyPyPy-3' half-sites separated by 0 ± 13 bp (EI-Deiry et al., 1992), by itself allows for many variations. In addition, analysis of promoter sequences transactivated by p53 showed that even the presence of several bases not conforming to the consensus is tolerated (Bargonetti et al., 1993; Foord et al., 1993). This amazing heterogeneity within p53 cognate sites asks for additional mechanisms providing speci®city and selectivity to sequence-speci®c transactivation by p53. Indeed, close examination of the various natural and arti®cial DNA sequences recognized by p53 demonstrated that, despite their sequence-heterogene- Dual role model for p53 function N Albrechtsen et al ity, many of them display an internal symmetry as a common feature. Internal symmetry is a hallmark of DNA sequences able to assume a non-B-DNA conformation upon topological stress. We therefore speculated whether p53 promoter DNA might assume dierent conformational states, and whether p53 might be able to interact with such DNA. Using a variety of arti®cial DNA substrates displaying the same p53 target sequence in dierent conformational states, we were able to demonstrate that p53 indeed is able to recognize isoforms of the same target sequence presented in dierent conformations. Interestingly, recognition of such isoforms strongly depended on p53 protein conformation: whereas recognition of isoforms in duplex B-DNA conformation required activation of p53 by addition of PAb421, recognition of isoforms in non-B-DNA conformations did not. On the contrary, binding of p53 to some of such isoforms was inhibited by PAb421. We concluded that sequencespeci®c DNA binding of p53 can be modulated both by p53 protein conformation and by the conformation of its cognate DNA sequence (Kim et al., 1997). This ®nding opens the way for a better understanding of target selectivity of p53 sequence-speci®c transactivation, as it is becoming increasingly accepted that chromatin con®guration, speci®cally promoter DNA conformation, is an important determinant of promoter activation. Accordingly, the promoter DNA can be present in an active conformation, which allows transcription, and an inactive conformation, which prohibits transcription. We suggest that p53-reactive promoters can adopt such conformations, and that promoter DNA topology and p53 protein conformation (and/or interaction with auxiliary proteins) determine the interaction of p53 with target gene promoters. In support of this model, we were able to show that e.g. recognition of the mdm2-promoter by p53 is impaired, when this promoter assumes a non-B DNA conformation in superhelical DNA. Relaxation of superhelicity by topoisomerase treatment restored p53 binding and increased the level of sequence-speci®c transactivation by p53 (Kim et al., submitted). In vivo, conformational changes in chromatin are mediated by proteins modeling chromatin architecture (Nickerson, 1998; Zlatanova and van Holde, 1998). With regard to our model it therefore may be more than coincidence that p53 interacts with at least two proteins involved in such processes, HMG1 (Jayaraman et al., 1998), and topoisomerase I (Gobert et al., 1996). Another protein that is involved in chromatin remodeling and p53 binding is the transcriptional co-activator CBP/p300. This protein forms a complex with p53, which in turn is able to enhance transcription of p53-dependent genes (Avantaggiati et al., 1997; Gu and Roeder, 1997). The interaction of CBP and p53 is strongly stimulated after phosphorylation of p53 at serine 15 (Lambert et al., 1998). CBP/p300 bears an intrinsic histone acetyltransferase (HAT) activity that is thought to be involved in histone acetylation and chromatin decondensation (Bannister and Kouzarides, 1996; Ogryzko et al., 1996). The HAT activity of CBP/p300 is controlled by cell cycle-dependent kinases and the oncoprotein E1A (Ait-Si-Ali et al., 1998). The HAT activity also phosphorylates p53 in vitro and thereby increases its transcriptional potential (Sakaguchi et al., 1998). HATmediated acetylation of p53 seems also to occur in vivo, particularly in response to DNA damage (Liu et al., 1999). Hence it appears that there exists another regulatory circuit that consists of HATs, p53, and the reorganization of the chromatin structure, that may respond to physiological situations such as the ongoing cell cycle and also to pathological threats, such as DNA damage. DNA repair after activation of p53 by DNA damage As outlined above, activation of p53 after DNA damage leads to a p53-induced growth arrest or apoptosis. Whereas induction of apoptosis bypasses any need to repair DNA damage, a p53-induced transient growth arrest will ful®ll its intention only, if it achieves an accurate repair of the damaged DNA. As sequence-speci®c transactivation of p53 target genes is regarded as the major function of p53 after its activation, upregulation of genes involved in DNA repair might be envisioned as a ®rst step to initiate repair of DNA damage. However, with the exception of the gadd45 gene (Kastan et al., 1992), and the p48 xeroderma pigmentosum gene (Hwang et al., 1999), which have been linked to DNA repair, no such direct eect of p53 is known. One thus has to conclude that DNA repair during a p53-induced growth arrest occurs in a p53-independent manner, or alternatively, that p53 participates in DNA repair by other activities. Although there is de®nitely p53independent DNA repair (as can be demonstrated in p53-de®cient cells), there is also evidence for a participation of p53 in repair processes, and for a p53-dependent activation of DNA repair. The assumption of a direct involvement of p53 in DNA repair leads to the prediction that mutations in p53 or the lack of p53 should lead to a defect in DNA repair. If p53 actively participates in DNA repair, p53 should interact with proteins that are part of DNA repair pathways and/or possess biochemical activities by which it could be part of repair pathways. To test for a direct involvement of p53 in DNA repair, Ford and Hanawalt examined Li-Fraumeni ®broblasts which were homozygous for a mutated p53 and found that these cells were indeed de®cient in the rate and extent of nuclear excision repair (NER) of genomic DNA (Ford and Hanawalt, 1995, 1997). In contrast, they did not observe a defect in transcriptioncoupled NER. This point, however, remains somewhat controversial, since other investigators found a de®ciency in transcription-coupled NER (Mirzayans et al., 1996; Wang et al., 1995). Reduced NER was also observed when the normal p53 function was disrupted by targeting p53 for degradation by the human papillomavirus E6 oncogene, or by expression of a dominant-negative mutant p53 (Smith et al., 1995). Furthermore, Li-Fraumeni-Syndrome cells with mutated p53 are impaired in their recovery for both RNA and DNA synthesis after UV-treatment, an anomaly which they share with the DNA repair disorders xeroderma pigmentosum and Cockayne's syndrome (Mirzayans et al., 1996). In this respect, p53 behaves like other proteins that are directly involved in DNA repair. But not only does a defect in p53 function result in a defective DNA repair, the induction of p53 after a 7709 Dual role model for p53 function N Albrechtsen et al 7710 treatment mimicking UV-damage even leads to an enhancement of DNA repair (Eller et al., 1997). Association of p53 with components of repair pathways Data showing a speci®c interaction of p53 with components of repair pathways provided clues as to how p53 is involved in DNA repair. Most of these proteins are members of the transcription factor IIH (TFIIH) multiprotein complex, which initiates basal transcription of RNA polymerase II and couples transcription with NER) (Leveillard et al., 1996; Wang et al., 1995; Xiao et al., 1994). p53 interacts with three components (XPB=ERCC3, XPD=ERCC2 and p62) of the TFIIH protein complex. p53 inhibits the helicase activity of XPB and XPD, probably through its strand-reannealing properties (Leveillard et al., 1996; Wang et al., 1995). The eect of TFIIH on p53 function is not yet known. It is possible that the interaction of TFIIH components with p53 serves to colocalize p53 at sites of DNA repair or transcriptional initiation. p53 also binds to Cockayne syndrome B protein (CSB), which was identi®ed as a repair protein by phenotypic complementation of an excision repairde®cient rodent cell line (Troelstra et al., 1990). The encoded protein is functionally defect in human Cockayne syndrome type B, the most common form of this disease. Aicted individuals are mentally retarded and sensitive to sunlight. Wang and colleagues were able to show that in vitro translated CSB also binds speci®cally to GST-wild type p53 fusion protein (Wang et al., 1995). CSB, like ERCC2 and ERCC3 also possesses an unwinding activity. Therefore, binding of p53 to helicases involved in NER (and possibly their inhibition) seems to be a common feature of p53, although no mechanistic explanation has been provided so far. Another p53-binding partner involved in DNA repair is the single-stranded DNA binding protein RPA. UV irradiation disrupts the RPA-p53 complex, correlating with an activation of p53, but only in cells which are capable to carry out global nucleotide excision repair (Abramova et al., 1997). Since UV-irradiation is a major signal for the activation and induction of p53, it seems likely that binding of p53 to RPA is involved in upstream regulation of p53-dependent damage response. Combined with the fact that a defect in p53 function leads to a defect in global NER, it may be more than coincidence that UV-irradiation leads to a disruption of the binding of RPA to p53 only in cells capable of global NER. A plausible model is that RPA in nonirradiated cells sequesters p53, but releases it after DNA damage. The released p53 could then act as a transcriptional activator, or/and could directly participate in global NER. Biochemical activities of p53 related to DNA repair An important feature of p53 in considering its direct involvement in DNA repair is its ability to interact with DNA in many dierent ways: p53 binds to double-stranded and single-stranded DNA in a nonsequence-speci®c manner (Steinmeyer and Deppert, 1988), to ends of double-strand breaks (Bakalkin et al., 1995), to Holliday junctions (Lee et al., 1997), and to DNA mismatches leading to DNA-`bulges' (Lee et al., 1995). These activities are important for p53's ability to bind to damaged DNA. p53 thus can `sense' and bind strongly to DNA damaged by ionizing radiation (Reed et al., 1995) and form highly stable complexes with insertion/deletion mismatches (Lee et al., 1995). p53 in this respect shows parallels to the DNA repair factor MSH2. Binding to damaged DNA is mediated by the C-terminal domain of p53, which has been shown to be important for the regulation of p53 function. The `sensing' mechanism forms the basis for a model in which p53 via its C-terminus recognizes DNA damage. p53 then becomes activated for sequence-speci®c DNA binding and for transactivation of target genes, which then participate in and enhance DNA repair (Jayaraman and Prives, 1995). However, this model is confronted with a major problem: it is dicult to envision how p53 will bind tightly to damaged DNA with a half-life of more than 2 h (Lee et al., 1995), and at the same time will transactivate genes which in all likelihood are located in a distant part of the genome, i.e. megabases away from the DNA damage locus. In addition, as already indicated above, so far no p53 target genes have been identi®ed, which could account for the global repair defect when p53 is mutated. Therefore we suggest that p53, in addition to damage recognition, could be directly involved in DNA repair processes by several biochemical activities, namely by its non-sequencespeci®c DNA binding activity, its DNA reannealing activity, its ability to promote DNA strand transfer, and its 3'-45' exonuclease activity. Especially the p53 intrinsic 3'-45' exonuclease activity, localized within the p53 core domain (Mummenbrauer et al., 1996); could be an important player in repair activities of p53. Exonucleases are required for DNA replication, DNA repair and recombination and often enhance the ®delity of these processes. As mutant p53 is exonuclease de®cient (Mummenbrauer et al., 1996), and cells expressing a mutant p53 are defective in global NER (Ford and Hanawalt, 1995, 1997; Wang and Prives, 1995; Mirzayans et al., 1996), this correlation might point to a possible role of the p53 exonuclease activity in DNA repair. The various biochemical activities of p53 and its interactions with viral and cellular proteins are summarized and related to p53 structure in Figure 1. Involvement of p53 in control of homologous recombination Recombination processes are subject to complex surveillance mechanisms ensuring high ®delity of DNA repair and of genetic transmission during meiosis (Haber, 1997; Xu et al., 1997). Regulatory circuits must also exist in order to establish dierences in DNA exchange frequencies between distinct genomic loci during meiosis, and in mitotically growing cells, where homologous recombination must be suppressed by a factor of 1000. p53 accumulates and becomes functionally activated (Lutzker and Levine, 1996; Siegel et al., 1995) upon the introduction of double strand breaks (DSB) into DNA (Nelson and Kastan, 1994). DSB arise spontaneously due to errors in replication, recombination or mitosis and can be Dual role model for p53 function N Albrechtsen et al 7711 Figure 1 p53 landmarks. Roman numerals represent the ®ve regions of p53 that are conserved within p53 from all vertebrates. Known phosphorylation (P) and acetylation (Ac) sites are indicated. The vertical bars, clustered in the center of the p53 molecule, indicate amino acid residues mutated in human tumors (hot spots are identi®ed by amino acid number). Shown below and indicated by horizontal bars is the current information concerning various domains of p53 for biological activities, p53 DNA interactions, and p53-protein complex formation. Abbreviations: CK 2, casein kinase 2; CSB, Cockayne's syndrome B protein; DNA PK, DNA dependent protein kinase; NLS, main nuclear localization signal; RP-A, replication protein A; SV 40, Simian Virus 40; TAF, transcription activating factor; TBP, TATA-Box binding protein; TF, transcription factor; XPB, xeroderma pigmentosum B protein; XPD, xeroderma pigmentosum D protein induced experimentally by ionizing radiation, radiomimetic drugs, or by the introduction of restriction endonucleases. DSB trigger both repair associated and targeted recombination processes, a connection which in turn renders p53 a likely candidate for being a regulatory factor in DNA exchange processes. During the last years we (WiesmuÈller et al., 1996) and others (Mekeel et al., 1997; Bertrand et al., 1997; Honma et al., 1997) demonstrated that p53 suppresses spontaneous inter- and intrachromosomal homologous recombination events at least by one to two orders of magnitude. The same groups also demonstrated that transcriptional transactivation and cell cycle control activities are separable from p53's involvement in homologous recombination by comparing the activities of cancer-related p53 mutants and C-terminally truncated p53 variants in these processes (DudenhoÈer et al., 1999; BS Lopez, Paris, and SN Powell, personal communication). Inhibition of p53 recombination control by SV40 SV40 provides a suitable model system for probing recombination, taking advantage of the small chromatin-packaged viral genome, which is ampli®ed episomally, and which promotes high frequency exchange rates for easy detection of recombination events (WiesmuÈller et al., 1996; Jasin et al., 1985; Snapka et al., 1991; Kawasaki et al., 1994). The SV40 tumor antigen (T-Ag) was found to be the causative agent for the elevated recombination frequencies of cellular and viral DNAs as well as for the stimulation of the closely related gene ampli®cation events (WiesmuÈller et al., 1996; Perry et al., 1992; Ishizaka et al., 1995; Cheng et al., 1997). SV40 T-Ag targets p53 in SV40 infection and transformation by forming a tight complex with p53. A possible role for p53-T-Ag complex formation is the elimination of a function of p53 in the control of recombination. By comparing the recombination rates between SV40 genomes expressing a wild type T-Ag or a T-Ag containing a point mutation which speci®cally abolishes T-Ag-p53 interactions (WiesmuÈller et al., 1996), we found that recombination frequencies in cells expressing a T-Ag unable to bind p53 were reduced by at least one order of magnitude. Therefore we conclude that suppression of homologous recombination events by wild type p53 can be alleviated by complex formation with SV40 T-Ag (WiesmuÈller et al., 1996). Elimination of p53-mediated suppression of recombination by T-Ag might also explain the ®nding that immortalization of human ®broblasts after stable transformation with T-Ag increases chromosomal recombination. Other viral binding partners, such as HPV16 E6 (Mekeel et al., 1997; Havre et al., 1995), Dual role model for p53 function N Albrechtsen et al 7712 and possibly the genetically destabilizing Hepatitis B virus (HBV) X antigen (Feitelson et al., 1993), may also neutralize the inhibitory function of p53 in recombination, thereby allowing a correlation between the targeting of p53 by these proteins and elevated recombination. Mismatch-recognition: A speci®c role for p53 in recombination control? We recently discovered that p53 inhibits DNA exchange most dramatically upon recognition of certain mismatches within nascent recombination intermediates (Dudenhoeer et al., 1998). Parallels to the functions of classical mismatch repair factors in recombination arise, as the mammalian MutS counterpart, MSH2, like p53, binds to Holliday junctions (Alani et al., 1997) and abolishes DNA exchange between divergent sequences (De Wind et al., 1995). In support of our idea, p53 was shown to equally aect homologous recombination between direct and indirect repeat substrates, which allows the conclusion that p53 controls recombination at the stage of strand invasion (BS Lopez, personal communication). Therefore, it is tempting to speculate that p53 monitors the ®delity of recombination in concert with the mismatch repair system. MSH2 and p53 do not seem to perform structural but rather regulatory functions during DNAexchange processes. Consistent with the hypothesis of two surveillance pathways acting in parallel, the inactivation of both pathways causes a synergism in tumorigenesis, evidence for which was recently given by the analysis of Msh2 and p53 double knock-out mice (Cranston et al., 1997). Therefore, MSH2 and p53 must be considered to be members of dierent genetic epistasis groups involved in the regulation of recombination processes. The view is consistent with our biochemical data suggesting complementary ®delity control due to an opposing mismatch speci®city for MSH2-GT-binding protein complexes, which most eciently recognize G-T single base mispairings (Hughes and Jiricny, 1992), and for p53tetramers, which display highest anities for A ± G mismatches (Dudenhoeer et al., 1998). It remains to be established, whether p53 upon encountering mismatches in heteroduplices transmits signals to downstream molecules, such as p21waf1, or/and blocks the progress of DNA exchange, possibly even by participating in error removal through its 3'-45' exonuclease activity. First indications for a p53mediated signal transduction mechanism coupled to recombination processes can be derived from the fact that the hRad51-complex partners Brca1 and Brca2 physically and genetically interact with p53 (StuÈrzbecher et al., 1996; Ouchi et al., 1998; Zhang et al., 1998; Lim and Hasty, 1996; Hakem et al., 1997; Ludwig et al., 1997), and that Brca1 seems to stimulate p53-dependent transcription, whereas overexpressed Brca2 and hRad51 display antagonistic eects (Marmorstein et al., 1998). In line with this proposal is the observation that puri®ed multiprotein complexes performing homologous recombination in vitro contain DNA polymerase e, DNA ligase and low levels of 5'-43' and 3'-45' exonuclease activities (Jessberger et al., 1993). Is there a role for p53 in DNA repair in its non-induced state? The transactivation and the exonuclease function of p53 seem to be mutually exclusive The active participation of p53 in DNA repair processes might not be restricted to situations activating a p53 response, like DNA damage in¯icted by exogenous factors. On the contrary, expanding p53's role as `guardian of the genome' by endowing it with a basic function in DNA repair processes in its non-induced state instead of being just a nonfunctional protein should greatly enhance its possibilities to preserve the integrity of the genome. Actually, some inconsistencies regarding the role of the waf1 gene as a major target in the p53 response to DNA damage already point to such a basic function. If indeed p53-mediated induction of the waf1 gene, and the ensuing growth arrest and stop of PCNAdependent DNA replication, would play a major role in p53's activities to ensure repair of DNA lesions, one would have to postulate that waf1 nullizygote mice should display a similar phenotype regarding tumor predisposition as p53 null mice. However, this is not the case, despite the ®nding that cells from waf1 nullizygote mice have largely lost the ability to arrest in G1 after DNA damage (Deng et al., 1995). Therefore, we assume that some other function of p53 is preventing the accumulation of mutations in these mice. We propose that such a function could be the `basic function' of p53, preventing the accumulation of mutations by its involvement in the repair of endogenous DNA damage. With the exception of sequence-speci®c transactivation of p53 target genes, which seems to be dependent on post-translational modi®cation of p53 after DNA damage, all of its other activities outlined above could, in principle, be exerted also by a non-induced p53. Possible limitations would only arise from the low amounts of p53 usually present in normal cells. However, even this argument is questionable, as it has been demonstrated that the transactivation function of p53 can be induced without a detectable rise in p53 levels (Hupp et al., 1995). Although there is no in vivo evidence for such a `basic' function of p53, comparative analysis of the major biochemical activities exerted by the p53 core domain, sequence-speci®c DNA binding and 3'-5' exonuclease activity (Figure 1), prompted us to suggest a direct participation of p53 in the repair of endogenous DNA damage, in preventing faulty DNA repair, and possibly also in DNA replication. As it seems rather unlikely that p53 will act as an exonuclease and simultaneously bind to DNA in a sequence-speci®c manner, the localization of the 3'-45' exonuclease activity of p53 to the same domain that exerts sequence-speci®c DNA binding poses the problem of how these activities are regulated. During deletion mapping analyses we found that C-terminal truncation of the C-terminal thirty amino acids of the p53 molecule activated its exonuclease activity by about a factor of ten, indicating that the C-terminal regulatory domain of p53 negatively in¯uences the p53 exonuclease activity. The same, however, has been shown for the sequence-speci®c DNA binding activity Dual role model for p53 function N Albrechtsen et al of p53, underscoring the importance of the p53 Cterminus for the regulation of p53 functions. Yet, whereas both activities were negatively regulated by the p53 C-terminus, treatments which aected the regulation of p53 activities by the p53 C-terminus had opposing eects on sequence-speci®c DNA binding and the exonuclease activity of p53: treatments, which activated sequence-speci®c DNA binding of p53, like addition of the monoclonal antibody PAb421 which binds to an epitope within the C-terminal regulatory domain, or enhancing the phosphorylation status of p53, strongly inhibited the p53 exonuclease activity (Janus et al., 1999). This ®nding points to the exciting possibility that p53 can exist in at least two dierent functionally active states. Since activation of sequencespeci®c DNA binding is considered to be a hallmark of p53 activation after DNA damage, we conclude that p53 looses its exonuclease activity when the sequencespeci®c DNA binding of p53 becomes activated after DNA damage. Conversely, however, one can also conclude that non-activated p53 exerts exonuclease activity, implying that a non-activated p53 is not equal to a non-functional one. Possible functions of the p53 exonuclease activity What could be the role of the p53 exonuclease activity in DNA repair? Exonucleases are required for nearly all processes of DNA metabolism, such as DNA replication, long patch DNA repair, postreplicative mismatch repair, and DNA recombination. An important type of error avoidance mechanism requiring exonuclease activities is the mismatch repair pathway. In bacteria this system invokes the coordinated action of the MutSLH damage recognition/ endonuclease complex along with the UvrD helicase, DNA polymerase III, DNA ligase, single-strand DNA binding protein, and any one of the exonucleases ExoI(3'-45' exo), ExoVII (3'-45' and 5'-43' exo) or RecJ (5'-43' exo) (Modrich, 1987, 1989). An involvement of p53's exonuclease in the postreplicative mismatch repair pathway would be of particular interest because this would link one of the enzymological functions of p53, i.e., its exonuclease activity, to the tumor suppressor function of the mammalian MutSLH homologues hMSH2, hMLH1, hPMS1, and hPMS2. A role complementary to these mismatch recognition proteins has already been proposed for p53 in mismatch recognition during recombination events (see above). Functional loss of either of these proteins leads to an increased incidence of colon carcinomas (for reviews see Bodmer et al., 1994; Modrich, 1994; Radman and Wagner, 1993). Furthermore, mutations in any of these genes display a generalized increase in spontaneous mutation rates, a replication error positive (RER+) phenotype, and a resistance to alkylating agents (Bhattacharyya et al., 1994; Leach et al., 1993; Parsons et al., 1993). In this respect, it might be more than coincidence that p53-de®cient animals show a relative resistance to alkylating agents (Harvey et al., 1993). Moreover, there is an inverse correlation between RER+ status and p53 mutation in colorectal cancer cell lines (Cottu et al., 1996), pointing to a synergistic action of gene products involved in mismatch repair with p53. This synergism is further corroborated by the ®nding that male mice nullizygous for both Msh2 and p53 develop tumors signi®cantly earlier than either of the single mutants (Cranston et al., 1997). Furthermore, there is some evidence that, like the p53 protein itself (see above), an intact mammalian MutSHL system displays an `anti-recombinogenic' eect (Fishel and Kolodner, 1995). In line with the possibility of a direct involvement of p53 in mismatch repair is the recent demonstration that wt p53 can `sense' DNA mismatch lesions by tightly binding to DNA containing insertion/deletion mismatches (Lee et al., 1995). Another possibility is p53's involvement in error avoidance by contributing a certain type of `proofreading' activity. There are at least six dierent cellular DNA polymerases designated as DNA polymerases a, b, g, d, e, and z that are involved in dierent aspects of DNA synthesis. Two of these, namely DNA polymerases a and b, are devoid of a 3'-45' exonuclease activity that excises mismatched nucleotides immediately after the incorporation step. Therefore, DNA polymerases a and b are particularly error prone and until now it is not known, how mismatched nucleotides incorporated by these two polymerases are removed (Kunkel, 1992). Moreover, both polymerases have a strong tendency to introduce frame-shift mutations by misinsertion or deletion of nucleotides (Huang, 1998). Since p53 recognizes DNA bulges caused by insertion/ deletion mismatches (Lee et al., 1995), it might be particularly suited to excise them via its 3'-45' exonuclease. Last but not least, p53 might act as a direct proofreader for the proofreader de®cient DNA polymerases a and b. A model for p53 function in the maintenance of genetic integrity by DNA repair Dierent subclasses of p53 may perform dierent functions within the same cell An important aspect for evaluating the activities of p53 in its induced and in its non-induced state is that not necessarily all p53 molecules must be in the same functionally active state, i.e. must exert the same function. Functional heterogeneity of multifunctional regulatory proteins is well known. Probably one of the best documented examples is the SV40 T-Ag, the major regulatory protein of SV40. Analyses of its subcellular localization and of its biochemical activities in SV40 lytically infected cells clearly demonstrated the coexistence of several functionally dierent subclasses of TAg molecules, involved in various aspects of viral replication (reviewed in Deppert and Schirmbeck, 1995). Assuming a similar situation for the multifunctional p53 molecule, one could envision that even after DNA damage only a certain fraction of the p53 molecules becomes activated for sequence-speci®c transcription, whereas other molecules remain in their non-induced state. Speci®city for selective activation (or non-activation) of p53 subclasses can be provided by subcellular compartmentalization of the p53 protein and/or by its association with dierent cellular proteins. The existence of functionally dierent subclasses of p53 within the same cell so far has not been proven. However, previous experiments from this laboratory at least demonstrated the presence of p53 in 7713 Dual role model for p53 function N Albrechtsen et al 7714 dierent nuclear compartments (Deppert and Haug, 1986). Therefore, it is possible that the p53 population not engaged in transcriptional regulation could exert functions other than induction of growth arrest or apoptosis, and directly participate in processes of DNA repair via its various biochemical activities described above. The exonuclease activity, for example, could be involved in repair processes such as DSB repair, which is thought to require DNA helicases, like the Ku autoantigen (Suwa et al., 1994; Tuteja et al., 1994), and exonucleases. Furthermore, p53 might act as an external proofreader for errors produced by cellular DNA polymerases involved in DNA replication and DNA repair also under DNA damage conditions. DNA polymerase a is certainly involved in nuclear DNA replication (Wang, 1991), but also has some function in DNA repair (Saxena et al., 1990; Oda et al., 1996; Holmes and Haber, 1999), whereas DNA polymerase b has been solely assigned to DNA repair processes (Wood and Shivji, 1997). When DNA damage has occurred, there is an apparent shut-o of PCNA-dependent DNA replication (Wood and Shivji, 1997; Flores-Rozas et al., 1994). Although PCNAdependent DNA-repair synthesis still might continue under conditions of p21waf1 induction (Li et al., 1994a, see above), DNA-repair synthesis could also be performed by the PCNA-independent and proofreader-free DNA polymerases a and/or b, for which p53 could provide the proof-reader function. If one assumes that after damage dierent functional subclasses of p53 will exist within the same cell, then the ensuing increase of p53 protein levels not only will activate the potential of p53 to transcribe p53 target genes, leading to growth arrest and to shut-o of PCNA-dependent DNA replication, but will also increase the amount of p53 with a 3'-45' proofreading exonuclease activity. Such p53 then could enhance the accuracy of DNA repair synthesis performed by the more error-prone DNA polymerases a and b. The `dual role' model for p53 The major conclusion which can be drawn from the above considerations is that the current picture of p53 as a guardian of the genome solely activated by cellular stress and/or DNA damage will be an oversimpli®cation. We assume that a non-induced p53 is not a protein without function. p53 in its non-induced state at least performs exonuclease activity, which could be involved in a variety of possible functions of p53 in DNA repair which all contribute to avoid mutations in the genome, as outlined above. We consider that the basic function of p53 is the repair of endogenous DNA damage, and the prevention of mutational events resulting from such damage. Superimposed are the up to now better characterized functions of p53 as a superior control element in integrating cellular stress signals, followed by the induction of either a growth arrest or of apoptosis. This model of a dual role of p53 as a guardian of the genome is outlined in Figure 2. If induction of p53 will activate only a fraction of p53 for sequence-speci®c transcription, the `basic function' of p53 must not be restricted to p53 in its non-induced state, and other, non-induced subclasses of p53 could directly engage in the various repair processes outlined Figure 2 Dual role of p53 as guardian of the genome. The current view of p53 as a superior control element in the responses to various types of cellular stress is depicted in the right half of the ®gure (adapted from Hall et al., 1996). The left half provides an extension of this model, according to which a non-induced p53 actively participates in the prevention of mutations arising from endogenous DNA damage. Note that functions of a non-induced p53 can also operate under conditions of cellular stress, if one assumes that after cellular stress dierent functional subclasses can exist within the same cell (for details see text) Dual role model for p53 function N Albrechtsen et al above. Thereby p53, after DNA damage then would be able to exert its full range of possible biochemical activities. Clearly, this model is highly speculative, but testable. For example, the postulated proofreader activity of p53 predicts a functional association of an exonucleaseactive p53 with the proofreader de®cient polymerases a and/or û. Biosensor studies indeed suggest that p53 binds to both polymerases with considerable anity (KuÈhn et al., 1998), and a speci®c complex between p53 and an immunologically distinct subclass of polymerase a could be detected in vivo (I Dornreiter, unpublished observation). Moreover, pol a can be anity-puri®ed together with p53 from monoclonal antibody columns directed against pol a (F Grosse, unpublished observations). 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