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Highly purified p53 protein from different sources was able to degrade DNA with a 3'-to-5' polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV-cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a "guardian of the genome."

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Recently, we described a new biological function of p53 in inhibiting recombination processes when encountering mismatches in heteroduplexes (DudenhoÈ er et al., 1998). Here, we characterized protein domains of p53 participating in this process by in vitro analysis of mutated p53 proteins, and by applying our SV40-based assay system on monkey cells, which express di erent p53 variants. We present evidence that both binding of arti®cial recombination intermediates and p53-dependent recombination control require an intact p53 core and the oligomerization domain, strongly suggesting that the recognition of DNA undergoing recombination represents an essential step of this genomic surveillance mechanism. Further analyses indicated a role of the C-terminus in negatively regulating recombination control, an e ect which can be neutralized by concurrent mismatch recognition. p53 lacking the oligomerization domain totally lost its ability to suppress homologous recombination. The cancer-related mutant p53(273H) was also signi®cantly defective in this function, although we observed only twofold reductions in the corresponding transactivation activities on p53-response elements in episomal constructs. HDM2, an inhibitor of p53's transcriptional and growth regulatory activities, interfered with the inhibition of DNA exchange processes by p53 only weakly. Thus, functions of p53 in recombination control can be structurally dissociated from p53-dependent transcriptional transactivation.

The archetypal human tumor suppressor p53 is considered to have unique transactivation properties. The assumption is based on the fact that additionally identified human p53 isoforms lack transcriptional activity. However, we provide evidence for the existence of an alternatively spliced p53 isoform (Deltap53) that exerts its transcriptional activity independent from p53. In contrast to p53, Deltap53 transactivates the endogenous p21 and 14-3-3sigma but not the mdm2, bax, and PIG3 promoter. Cell cycle studies showed that Deltap53 displays its differential transcriptional activity only in damaged S phase cells. Upon activation of the ATR-intra-S phase checkpoint, Deltap53, but not p53, transactivates the Cdk inhibitor p21. Induction of p21 results in downregulation of cyclin A-Cdk activity and accordingly attenuation of S phase progression. Data demonstrate that the Deltap53-p21-cyclin A-Cdk pathway is crucial to facilitate uncoupling of repair and replication events, indicating that Deltap53 is an essential element of the ATR-intra-S phase checkpoint.

We demonstrate that wild-type p53 inhibits homologous recombination. To analyze DNA substrate specificities in this process, we designed recombination experiments such that coinfection of simian virus 40 mutant pairs generated heteroduplexes with distinctly unpaired regions. DNA exchanges producing single C-T and A-G mismatches were inhibited four-to sixfold more effectively than DNA exchanges producing G-T and A-C single-base mispairings or unpaired regions of three base pairs comprising G-T/A-C mismatches. p53 bound specifically to three-stranded DNA substrates, mimicking early recombination intermediates. The K D values for the interactions of p53 with three-stranded substrates displaying differently paired and unpaired regions reflected the mismatch base specificities observed in recombination assays in a qualitative and quantitative manner. On the basis of these results, we would like to advance the hypothesis that p53, like classical mismatch repair factors, checks the fidelity of homologous recombination processes by specific mismatch recognition.p53 germ line mutations are associated with a deficit to maintain genomic stability along with an increase of spontaneous gene amplification rates (17,52,93), thereby accelerating the multistep process of tumor progression (81). This phenotype has been explained by the loss of p53 cell cycle checkpoint control (38,39,46). DNA damage (38, 39, 60) and suboptimal growth situations, such as an increase of oxygen radicals (28) or ribonucleotide depletion (50), are signals for p53-mediated accumulation and functional activation (54,68). Depending on the cell type, p53 induces cell cycle arrest or apoptosis predominantly via transcriptional transactivation of genes coding for the cyclin-dependent kinase inhibitor p21/WAF1/CIP1/SDI1 (21, 23) or the apoptotic factor Bax (56). As a consequence, cells are unable to replicate their DNA under conditions which may lead or may have led to chromosome breaks (3), thereby preventing the manifestation and aggravation of genomic lesions in S phase. Strikingly, the same molecular signal triggering the DNA damage response by p53, namely, DNA strand breaks (60), also initiates V(D)J recombination (79), meiotic recombination (27), recombination repair (75), and gene amplification (19) events. There is evidence for an at least indirect involvement of p53 in V(D)J recombination, as ␥ irradiation can rescue rearrangement at multiple T-cell receptor loci by a p53-dependent bypass mechanism in scid mice (2, 12). A role for p53 in meiotic recombination has been postulated from the observation that p53 mRNA expression in testes of mice is high and specific for spermatocytes in zygotene to pachytene, the meiotic stages at which homologous chromosomes synapse for genetic exchange (65, 69). Intriguingly, the mitotic checkpoint factor Atm, the product of the gene mutated in patients with ataxia telangiectasia (66), is also found in spermatocytes of meiosis I. Atm belongs to the family of phosphatidylinositol 3-kinase-like protein kinases which, li...

. Thus, TGF-␣ is able to promote progression throughout G 1 , but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-␣ transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.

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