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...

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominantoncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT ''DNA-unwinding motif,'' which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MARderived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GCclamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.

The mutational effects at the mRNA level were investigated by RT-PCR analysis of nine different nonsense mutations (Q39X, E60X, R75X, G542X, L719X, Y1092X, R1162X, S1196X, W1282X) and one frameshift mutation (1078delT) within the CFTR gene. With the exception of mutation R1162X, reduced mRNA levels ranging from 30% to less than 5% of the wild type have been observed. In case of the R75X and E60X mutations, the mRNA reduction was accompanied by the appearance of atypical CFTR isoforms. Single exon 3 skipping, as well as joint exon 2 and 3 skipping, was observed in lymphocyte and nasal epithelial mRNA derived from R75X alleles. The analysis of mRNA transcribed from E60X alleles revealed skipping of exon 3 (lymphocytes and nasal epithelial cells) or skipping of exons 3 and 4 (nasal epithelial cells). With the exception of the E60X mutation, no obvious tissue-specific differences in the splicing pattern and ratios of mutation to wild-type transcripts were detected between lymphocytes and nasal epithelial cells. In addition to aberrant splicing, the reduction of transcripts is the most common effect of nonsense and frameshift mutations within the CFTR gene.