Genetic stabilization by p53 involves growth regulatory and repair pathways

p53 performs a plethora of activities, which are directed towards the maintenance of the genomic integrity and constitute its universal role as a tumor suppressor. 1000 to 10000 latent p53 molecules are permanently available in order to monitor DNA exchange processes in mitotically growing cells. After the introduction of major DNA injuries the levels of posttranslationally modified p53 proteins rise, which in turn transcriptionally signal transient cell cycle arrest or apoptotic cell death, depending on the extent of damage. Taken together, p53 inhibits the manifestation of genomic instabilities at different control levels both during naturally occurring metabolic processes and in response to genotoxic treatments.

p53 performs a plethora of activities, which are directed towards the maintenance of the genomic integrity and constitute its universal role as a tumor suppressor. 1000 to 10000 latent p53 molecules are permanently available in order to monitor DNA exchange processes in mitotically growing cells. After the introduction of major DNA injuries the levels of posttranslationally modified p53 proteins rise, which in turn transcriptionally signal transient cell cycle arrest or apoptotic cell death, depending on the extent of damage. Taken together, p53 inhibits the manifestation of genomic instabilities at different control levels both during naturally occurring metabolic processes and in response to genotoxic treatments.

GENOMIC STABILIZATION VIA CHECKPOINT CONTROL
Loss of p53 function by either mutation, nuclear exclusion, complex inactivation, or accelerated degradation via the newly discovered ARF-MDM2 pathway are observed in the majority of human tumors [1,2]. The results of extensive research efforts have indicated that cell cycle control and the initiation of apoptotic cell death by p53 represent important pathways to suppress genomic instabilities, thereby preventing tumorigenesis [3,4]. p53-dependent checkpoint functions are triggered by DNA strand breaks introduced either directly, e.g., via ionizing irradiation, or indirectly, after the conversion of DNA adducts by DNA repair or replication [5]. An alternative signal transduction mechanism involving p53 seems to emanate from stalled RNA polymerases, e.g., after UV irradiation [6]. DNA damage activates p53 through posttranslational modifications by specific kinases, such as the strand break sensor Atm, acetylases, and poly(ADP-ribose)polymerase, which prevent proteolysis and enhance binding of p53 to consensus sequences within the genome [7][8][9]. Among the products of p53 target genes, the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 is essential for the execution of cell cycle arrest at the G1/S transition and to sustain a G2 arrest under certain circumstances [4,10]. The product of a 14-3-3 gene, which is also transcriptionally activated by p53, was reported to inactivate the protein phosphatase Cdc25C by sequestration [11]. Cdc25C is required for the activation of Cdc2 kinase at the G2/M checkpoint. Contrary opinions exist on a possible role of the p53responsive gene GADD45 in excision repair [12][13][14]. Meanwhile, functions of GADD45 in chromatin remodeling and of GADD45-p21 complexes in cell cycle regulation were proposed [15,16]. Apoptotic signaling involves transcriptionindependent pathways [17] and the activation of target genes, such as bax and IGF-BP3, encoding antagonists of Bcl-2 and insulin-like growth factor-1, respectively [4].
From these observations, it has been concluded that genomic stabilization and tumor suppression by p53 rely on cell cycle arrest at the G1/S transition, which prevents the manifestation of unrepaired chromosome alterations, on cell cycle arrest at the G2/M transition, which inhibits the distribution of defective genomes, and on the initiation of apoptosis after the introduction of irrepairable damage. This view is in agreement with the phenotype of p53/mice, which accumulate chromosomal aberrations and suffer from fatal tumors within 6 months [18]. However, p21/mice do not show increased cancer susceptibilities [19], raising the possibility that activities of p53, other than those related to growth control, might contribute to the suppression of tumor formation. Even further, according to a recent report, p53 seems to retain tumor suppressor functions in mice after treatment with PFTα, a drug, which had been selected due to its properties to specifically inactivate p53-dependent transcription and apoptosis [20].

INVOLVEMENT IN DNA REPAIR
Ideas on an active participation of p53 in processes of the DNA metabolism were inspired by the discoveries of enzymatic activities, such as the reannealing of short DNA stretches [21,22] and the 3 to 5 exonuclease activity [23]. p53 also binds to the two helicase components, XPB and XPD, of the dual transcription initiation/repair factor TFIIH, to CSB, another helicase involved in nucleotide excision repair, and to the Werner's Syndrome Protein, a helicase and exonuclease with putative functions in DNA replication [4,24,25]. Several groups reported on defective nucleotide excision repair in cells lacking wild-type p53, as determined by the removal of pyrimidine dimers [26,27]. Others noticed an in- crease in sister chromatid exchanges after UV in cells from p53/mice rather than differences in excision repair [28]. The latter observation might point towards a role of p53 in UVirradiation induced DNA exchange events, since recombination is frequently coupled to DNA synthesis in order to allow the removal of unrepaired lesions, such as unexcised photoproducts. Indeed, using different test systems, several groups found that p53 suppresses spontaneous inter-and intrachromosomal homologous recombination events by at least one to two orders of magnitude [29][30][31][32]. p53 has also been shown to interact with proteins involved in homologous DNA recombination processes, namely the initial strand transferase Rad51 and the Rad51 complex partners BRCA1 and 2 [24,33]. With respect to the breast cancer susceptibility gene products BRCA1 and BRCA2, functions in DNA repair, in the assembly of ionizing radiation-induced Rad51 complexes, in cell cycle control via transcriptional regulation of p21 WAF1/CIP1 and in mediating apoptosis via GADD45 have been ascribed [34][35][36][37]. Concerning the mechanism underlying the control of homologous recombination events by p53, we suggested that p53 monitors the fidelity of strand exchange events [32]. SV40-virus based recombination assays in combination with in vitro binding studies unveiled qualitative and quantitative correlations between the binding affinities for heteroduplex joints with certain mismatches and the inhibition of DNA exchange events creating the corresponding DNA intermediates. Since homologous recombination processes are frequently associated with DNA synthesis, it is interesting to note that p53 was found to excise mispaired nucleotides from DNA in a polymerase α based in vitro replication assay [38]. Before p53, another tumor suppressor, MSH2, had already been described to counteract DNA exchange processes between divergent sequences beyond its central role in postreplicative mismatch repair [39]. In agreement with the idea that p53 and MSH2 perform complementary functions in controlling the fidelity of homologous recombination processes, mice nullizygous for both MSH2 and p53 display synergistically increased cancer susceptibilities [40].
The critical question, whether the control of spontaneous and radiation-induced homologous recombination processes is tied to p53's growth regulatory functions was answered unequivocally by three groups [41][42][43]: Analyses of cell lines, expressing either different p53 mutants or wildtype p53 together with the p53-antagonist HDM2, demonstrated that recombination control is performed independently of p53-functions in transcription and cell-cycle control. Furthermore, it was observed that small protein amounts are sufficient for the inhibition of recombination processes by p53, whereas growth-related functions are exerted in a dose-dependent manner. These findings support the dual role model (see Figure 1), which attributes distinct functions to p53 in its latent and in its activated state, respectively [24]. It is important to note that homologous recombination in mitotically growing cells is suppressed by a factor of 1000 as compared to meiotic recombination. This might explain why meiotic exchange rates are not further elevated by the loss of p53 functions [44]. On the other hand, elevated frequencies of Rad51-dependent recombination was observed to accompany cellular immortalization processes [45]. Con-sidering the experimental data, which describe the regulatory role of wild-type p53 in DNA exchange processes of mitotically growing cells [41][42][43], the surveillance of homologous recombination by p53 is a good candidate to play a role in restraining spontaneous DNA rearrangements. Consequently, p53 might prevent tumor formation both by functions in growth regulatory and in repair processes.