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...

In this study, we characterize the molecular and functional features of a novel protein called SPOC1. SPOC1 RNA expression was previously reported to be highest in highly proliferating tissues and increased in a subset of ovarian carcinoma patients, which statistically correlated with poor prognosis and residual disease. These observations implied that SPOC1 might play a role in cellular proliferation and oncogenesis. Here we show that the endogenous SPOC1 protein is labile, primarily chromatin associated and its expression as well as localization are regulated throughout the cell cycle. SPOC1 is dynamically regulated during mitosis with increased expression levels and biphasic localization to mitotic chromosomes indicating a functional role of SPOC1 in mitotic processes. Consistent with this postulate, SPOC1 siRNA knockdown experiments resulted in defects in mitotic chromosome condensation, alignment and aberrant sister chromatid segregation. Finally, we have been able to show, using micrococcal nuclease (MNase) chromatin-digestion assays that SPOC1 expression levels proportionally influence the degree of chromatin compaction. Collectively, our findings show that SPOC1 modulates chromatin structure and that tight regulation of its expression levels and subcellular localization during mitosis are crucial for proper chromosome condensation and cell division.

Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase ␣-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G 1 , whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G 2 . Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.The initiation of chromosomal DNA replication in eukaryotes can be divided into two major independent events (reviewed in references 5 and 10). The first event takes place during the G 1 phase, when a preinitiation complex is formed at the origin of replication. The complex formation requires the sequential binding of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance proteins (MCM). The assembly of MCM on the chromatin plays an important role in generating a replication-competent, licensed origin. The second event occurs during the G 1 /S transition, when cyclin-dependent protein kinases (Cdks) as well as the Cdc7/ Dbf4 kinase convert the preinitiation complex into an active replication fork by an unknown process. In addition, activation of the preinitiation complex at the G 1 /S transition requires sequential chromatin binding of the replication factors Cdc45, RP-A, and polymerase ␣-primase (Pol-Prim) (37). After the RP-A-dependent unwinding of replication origins (37), the essential initiator Pol-Prim is recruited to the unwound origin, most likely by specific protein-protein interaction with chromatin-bound Cdc45 and/or RP-A (1, 8, 19).Pol-Prim isolated from a wide range of phylogenetic species contains a similar set of four polypeptides. The enzyme complex is composed of a 180-kDa polypeptide containing the catalytic function; a polypeptide of about 70 kDa, which is thought to be the regulatory subunit; and two polypeptides of 58 and 48 kDa associated with primase activity (reviewed in reference 38). Pol-Prim is the only enzyme capable of initiating DNA synthesis de novo by first synthesizing an RNA primer and then extending the primer by polymerization to produce a short 30-nucleotide DNA extension, which yields an RNA-DNA primer of approximately 40 nucleotides in length (reviewed in reference 38). Subsequently, in an ATP-dependent process, RF-C initiates po...

During infection, simian virus 40 (SV40) attempts to take hold of the cell, while the host responds with various defense systems, including the ataxia-telangiectasia mutated/ATM-Rad3 related (ATM/ATR)-mediated DNA damage response pathways. Here we show that upon viral infection, ATR directly activates the p53 isoform ⌬p53, leading to upregulation of the Cdk inhibitor p21 and downregulation of cyclin A-Cdk2/1 (AK) activity, which force the host to stay in the replicative S phase. Moreover, downregulation of AK activity is a prerequisite for the generation of hypophosphorylated, origin-competent DNA polymerase ␣-primase (hypoPol␣), which is, unlike AK-phosphorylated Pol␣ (P-Pol␣), recruited by SV40 large T antigen (T-Ag) to initiate viral DNA replication. Prevention of the downregulation of AK activity by inactivation of ATR-⌬p53-p21 signaling significantly reduced the T-Ag-interacting hypo-Pol␣ population and, accordingly, SV40 replication efficiency. Moreover, the ATR-⌬p53 pathway facilitates the proteasomal degradation of the 180-kDa catalytic subunit of the non-T-Ag-interacting P-Pol␣, giving rise to T-Ag-interacting hypo-Pol␣. Thus, the purpose of activating the ATR-⌬p53-p21-mediated intra-S checkpoint is to maintain the host in S phase, an optimal environment for SV40 replication, and to modulate the host DNA replicase, which is indispensable for viral amplification.Infection of quiescent CV-1 cells with the primate polyomavirus simian virus 40 (SV40) induces cell cycle progression and stimulates host cell DNA replication, which is mandatory for viral amplification. SV40 uses only a single viral protein, T antigen (T-Ag), for its own replication; all other components have to be provided by the host. Initially, a specifically phosphorylated subclass of T-Ag binds to a palindromic sequence in the SV40 origin (43), and in the presence of ATP, T-Ag forms a double-hexamer nucleoprotein complex leading to structural distortion and unwinding of origin DNA sequences (5). In concert with the cellular single-strand DNA binding protein RPA and topoisomerase I, the DNA helicase activity of T-Ag promotes more-extensive origin unwinding, forming a preinitiation complex (pre-RC), resulting in an initiation complex (53). Once the initiation complex forms, the primase activity of the heterotetrameric DNA polymerase ␣-primase (Pol␣) complex, consisting of the p180 catalytic subunit, the p70 regulatory subunit, and the p48/58 primase subunits, synthesizes a short RNA primer on each template strand, which is extended by the DNA polymerase activity of Pol␣ (6, 17). Immediately after the first nascent RNA/DNA primer is synthesized, the complete replication machinery is assembled, and elongation at both forks by the processive DNA polymerase ␦ ensues (62). Thus, during the initiation of SV40 replication, T-Ag performs many of the functions attributed to the eukaryotic pre-RC complex proteins, including Orc, Cdc6, Cdt1, and kinase-independent cyclin E, which facilitates loading of the putative replication helicase Mcm2-7 onto the e...

Transcriptional activation by the tumor suppressor p53 is regulated at multiple levels, including posttranslational modi®cations of the p53 protein, interaction of p53 with various regulatory proteins, or at the level of sequencespeci®c DNA binding to the response elements in p53's target genes. We here propose as an additional regulatory mechanism that the DNA topology of p53-responsive promoters may determine the interaction of p53 with its target genes. We demonstrate that sequencespeci®c DNA binding (SSDB) and transcriptional activation by p53 of the mdm2 promoter is inhibited when this promoter is present in supercoiled DNA, where it forms a non-B-DNA structure which spans the p53-responsive elements. Relaxation of the supercoiled DNA in vitro resulted in conversion of the non-B-DNA to a B-DNA conformation within the mdm2 promoter, and correlated with an enhanced SSDB of p53 and an elevated expression of a reporter gene. In contrast, sequence speci®c DNA binding and transcriptional activation of the p21 promoter were not inhibited by DNA supercoiling. We propose that conformational alterations within p53-responsive sites, which either promote or prohibit sequence speci®c DNA binding of p53, are an important feature in orchestrating the activation of dierent p53 responsive promoters.

Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.

Inhibition of homologous recombination (HR) is believed to be a transactivation-independent function of p53 that protects from genetic instability. Misrepair by HR can lead to genetic alterations such as translocations, duplications, insertions and loss of heterozygosity, which all bear the risk of driving oncogenic transformation. Regulation of HR by wild-type p53 (wtp53) should prevent these genomic rearrangements. Mutation of p53 is a frequent event during carcinogenesis. In particular, dominantnegative mutants inhibiting wtp53 expressed from the unperturbed allel can drive oncogenic transformation by disrupting the p53-dependent anticancer barrier. Here, we asked whether the hot spot mutants R175H and R273H relax HR control in p53proficient cells. Utilizing an I-SceI-based reporter assay, we observed a moderate (1.5 Â) stimulation of HR upon expression of the mutant proteins in p53-proficient CV-1, but not in p53-deficient H1299 cells. Importantly, the stimulatory effect was exactly paralleled by an increase in the number of HR competent Sand G2-phase cells, which can well explain the enhanced recombination frequencies. Furthermore, the impact on HR exerted by the transactivation domain double-mutant L22Q/W23S and mutant R273P, both of which were reported to regulate HR independently of G1-arrest execution, is also exactly mirrored by cell-cycle behavior. These results are in contrast to previous concepts stating that the transactivation-independent impact of p53 on HR is a general phenomenon valid for replication-associated and also for directly induced double-strand break. Our data strongly suggest that the latter is largely mediated by cell-cycle regulation, a classical transactivation-dependent function of p53.