Doing the right thing: feedback control and p53.

Recent evidence suggests that exposure of cells to DNA-damaging agents causes a rise in the levels of the p53 tumor suppressor protein and arrest of progression through the cell cycle. p53 may therefore resemble a member of the RAD gene class identified in yeast, RAD9, which allows cells to repair DNA before continuation of the cell cycle. The evidence that p53 is a sequence-specific, DNA-binding protein that can regulate transcription suggests several ways in which p53 might effect this growth cessation.

Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes.

Oligonucleotides complementary to regions of U1 and U2 small nuclear RNAs (snRNAs), when injected into Xenopus laevis oocytes, rapidly induced the specific degradation of U1 and U2 snRNAs, respectively, and then themselves were degraded. After such treatment, splicing of simian virus 40 (SV40) late pre-mRNA transcribed from microinjected viral DNA was blocked in oocytes. If before introduction of SV40 DNA into oocytes HeLa cell U1 or U2 snRNAs were injected and allowed to assemble into small nuclear ribonucleoprotein particle (snRNP)-like complexes, SV40 late RNA was as efficiently spliced as in oocytes that did not receive U1 or U2 oligonucleotides. This demonstrates that oocytes can form fully functional hybrid U1 and U2 snRNPs consisting of human snRNA and amphibian proteins.

Identification of p53 as a sequence-specific DNA-binding protein.

The tumor-suppressor gene p53 is altered by missense mutation in numerous human malignancies. However, the biochemical properties of p53 and the effect of mutation on these properties are unclear. A human DNA sequence was identified that binds specifically to wild-type human p53 protein in vitro. As few as 33 base pairs were sufficient to confer specific binding. Certain guanines within this 33-base pair region were critical, as methylation of these guanines or their substitution with thymine-abrogated binding. Human p53 proteins containing either of two missense mutations commonly found in human tumors were unable to bind significantly to this sequence. These data suggest that a function of p53 may be mediated by its ability to bind to specific DNA sequences in the human genome, and that this activity is altered by mutations that occur in human tumors.

Enhanced phosphorylation of p53 by ATM in response to DNA damage.

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function?

Although still controversial, the presence of mutant p53 in cancer cells may result in more aggressive tumors and correspondingly worse outcomes. The means by which mutant p53 exerts such pro-oncogenic activity are currently under extensive investigation and different models have been proposed. We focus here on a proposed mechanism by which a subset of tumor-derived p53 mutants physically interact with p53 family members, p63 and p73, and negatively regulate their proapoptotic function. Both cell-based assays and knock-in mice expressing mutant forms of p53 support this model. As more than half of human tumors harbor mutant forms of p53 protein, approaches aimed at disrupting the pathological interactions among p53 family members might be of clinical value.

Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

The p53 tumor suppressor is a nucleocytoplasmic shuttling protein that is found predominantly in the nucleus of cells. In addition to mutation, abnormal p53 cellular localization is one of the mechanisms that inactivate p53 function. To further understand features of p53 that contribute to the regulation of its trafficking within the cell, we analysed the subnuclear localization of wild-type and mutant p53 in human cells that were either permeabilized with detergent or treated with the proteasome inhibitor MG132. We, here, show that either endogenously expressed or exogenously added p53 protein localizes to the nucleolus in detergent-permeabilized cells in a concentration- and ATP hydrolysis-dependent manner. Two discrete regions within the carboxyl terminus of p53 are essential for nucleolar localization in permeabilized cells. Similarly, localization of p53 to the nucleolus after proteasome inhibition in unpermeabilized cells requires sequences within the carboxyl terminus of p53. Interestingly, genotoxic stress markedly decreases the association of p53 with the nucleolus, and phosphorylation of p53 at S392, a site that is modified by such stress, partially impairs its nucleolar localization. The possible significance of these findings is discussed.

Correction: Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

Following the publication of this article the authors noted that two images were duplicated in Figure 2B. The corrected figure 2B is below. The authors wish to apologize for any inconvenience caused.

P21Cip1/WAF1 downregulation is required for efficient PCNA ubiquitination after UV irradiation.

p21(Cip1/WAF1) is a known inhibitor of the short-gap filling activity of proliferating cell nuclear antigen (PCNA) during DNA repair. In agreement, p21 degradation after UV irradiation promotes PCNA-dependent repair. Recent reports have identified ubiquitination of PCNA as a relevant feature for PCNA-dependent DNA repair. Here, we show that PCNA ubiquitination in human cells is notably augmented after UV irradiation and other genotoxic treatments such as hydroxyurea, aphidicolin and methylmethane sulfonate. Intriguingly, those DNA damaging agents also promoted downregulation of p21. While ubiquitination of PCNA was not affected by deficient nucleotide excision repair (NER) and was observed in both proliferating and arrested cells, stable p21 expression caused a significant reduction in UV-induced ubiquitinated PCNA. Surprisingly, the negative regulation of PCNA ubiquitination by p21 does not depend on the direct interaction with PCNA but requires the cyclin dependent kinase binding domain of p21. Taken together, our data suggest that p21 downregulation plays a role in efficient PCNA ubiquitination after UV irradiation.

p53 tumor suppressor protein regulates the levels of huntingtin gene expression.

The p53 protein is a transcription factor that integrates various cellular stress signals. The accumulation of the mutant huntingtin protein with an expanded polyglutamine tract plays a central role in the pathology of human Huntington's disease. We found that the huntingtin gene contains multiple putative p53-responsive elements and p53 binds to these elements both in vivo and in vitro. p53 activation in cultured human cells, either by a temperature-sensitive mutant p53 protein or by gamma-irradiation (gamma-irradiation), increases huntingtin mRNA and protein expression. Similarly, murine huntingtin also contains multiple putative p53-responsive elements and its expression is induced by p53 activation in cultured cells. Moreover, gamma-irradiation, which activates p53, increases huntingtin gene expression in the striatum and cortex of mouse brain, the major pathological sites for Huntington's disease, in p53+/+ but not the isogenic p53-/- mice. These results demonstrate that p53 protein can regulate huntingtin expression at transcriptional level, and suggest that a p53 stress response could be a modulator of the process of Huntington's disease.

Transcriptional regulation by p53: one protein, many possibilities.

The p53 tumor suppressor protein is a DNA sequence-specific transcriptional regulator that, in response to various forms of cellular stress, controls the expression of numerous genes involved in cellular outcomes including among others, cell cycle arrest and cell death. Two key features of the p53 protein are required for its transcriptional activities: its ability to recognize and bind specific DNA sequences and to recruit both general and specialized transcriptional co-regulators. In fact, multiple interactions with co-activators and co-repressors as well as with the components of the general transcriptional machinery allow p53 to either promote or inhibit transcription of different target genes. This review focuses on some of the salient features of the interactions of p53 with DNA and with factors that regulate transcription. We discuss as well the complexities of the functional domains of p53 with respect to these interactions.

A unique subpopulation of murine DNA polymerase alpha/primase specifically interacts with polyomavirus T antigen and stimulates DNA replication.

Murine cells or cell extracts support the replication of plasmids containing the replication origin (ori-DNA) of polyomavirus (Py) but not that of simian virus 40 (SV40), whereas human cells or cell extracts support the replication of SV40 ori-DNA but not that of Py ori-DNA. It was shown previously that fractions containing DNA polymerase alpha/primase from permissive cells allow viral ori-DNA replication to proceed in extracts of nonpermissive cells. To extend these observations, the binding of Py T antigen to both the permissive and nonpermissive DNA polymerase alpha/primase was examined. Py T antigen was retained by a murine DNA polymerase alpha/primase but not by a human DNA polymerase alpha/primase affinity column. Likewise, a Py T antigen affinity column retained DNA polymerase alpha/primase activity from murine cells but not from human cells. The murine fraction which bound to the Py T antigen column was able to stimulate Py ori-DNA replication in the nonpermissive extract. However, the DNA polymerase alpha/primase activity in this murine fraction constituted only a relatively small proportion (approximately 20 to 40%) of the total murine DNA polymerase alpha/primase that had been applied to the column. The DNA polymerase alpha/primase purified from the nonbound murine fraction, although far more replete in this activity, was incapable of supporting Py DNA replication. The two forms of murine DNA polymerase alpha/primase also differed in their interactions with Py T antigen. Our data thus demonstrate that there are two distinct populations of DNA polymerase alpha/primase in murine cells and that species-specific interactions between T antigen and DNA polymerases can be identified. They may also provide the basis for initiating a novel means of characterizing unique subpopulations of DNA polymerase alpha/primase.

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.

p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition.

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.

Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA.

Human replication protein A (RP-A) (also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. Potentially important to both these functions, it is also capable of complex formation with the tumor suppressor protein p53. Here we show that although p53 is unable to prevent RP-A from associating with a range of single-stranded DNAs in solution, RP-A is able to strongly inhibit p53 from functioning as a sequence-specific DNA binding protein when the two proteins are complexed. This inhibition, in turn, can be regulated by the presence of various lengths of single-stranded DNAs, as RP-A, when bound to these single-stranded DNAs, is unable to interact with p53. Interestingly, the lengths of single-stranded DNA capable of relieving complex formation between the two proteins represent forms that might be introduced through repair and replicative events. Increasing p53 concentrations can also overcome the inhibition by steady-state levels of RP-A, potentially mimicking cellular points of balance. Finally, it has been shown previously that p53 can itself be stimulated for site-specific DNA binding when complexed through the C terminus with short single strands of DNA, and here we show that p53 stays bound to these short strands even after binding a physiologically relevant site. These results identify a potential dual role for single-stranded DNA in the regulation of DNA binding by p53 and give insights into the p53 response to DNA damage.

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.

Poly(A) polymerase phosphorylation is dependent on novel interactions with cyclins.

We have previously shown that poly(A) polymerase (PAP) is negatively regulated by cyclin B-cdc2 kinase hyperphosphorylation in the M phase of the cell cycle. Here we show that cyclin B(1) binds PAP directly, and we demonstrate further that this interaction is mediated by a stretch of amino acids in PAP with homology to the cyclin recognition motif (CRM), a sequence previously shown in several cell cycle regulators to target specifically G(1)-phase-type cyclins. We find that PAP interacts with not only G(1)- but also G(2)-type cyclins via the CRM and is a substrate for phosphorylation by both types of cyclin-cdk pairs. PAP's CRM shows novel, concentration-dependent effects when introduced as an 8-mer peptide into binding and kinase assays. While higher concentrations of PAP's CRM block PAP-cyclin binding and phosphorylation, lower concentrations induce dramatic stimulation of both activities. Our data not only support the notion that PAP is directly regulated by cyclin-dependent kinases throughout the cell cycle but also introduce a novel type of CRM that functionally interacts with both G(1)- and G(2)-type cyclins in an unexpected way.

p73 function is inhibited by tumor-derived p53 mutants in mammalian cells.

The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator. Recent studies have identified a p53 relative, termed p73. We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73alpha and -beta isoforms to transactivate various p53-responsive promoters. We show that both p73alpha and p73beta activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299. However, a number of significant differences were observed between p53 and p73 and even between p73alpha and p73beta. Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37 degreesC. Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay. When we examined complex formation in transfected mammalian cells, we found that p73alpha coprecipitates with mutant but not wild-type p53. Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73. By cotransfecting p73alpha along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73alpha. This decrease in transcriptional activity is correlated with the reduced ability of p73alpha to promote apoptosis in the presence of tumor-derived p53 mutants. Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.

A mutant p53 that discriminates between p53-responsive genes cannot induce apoptosis.

Human wild-type (wt) p53 can induce apoptosis in transiently transfected H1299 cells maintained at 37 degrees C, whereas tumor-derived mutant forms of p53 (with the mutation Ala-143, His-175, or Trp-248) fail to do so. At 37 degrees C, p53 with a mutation to Ala at amino acid 143 (p53Ala143) was transcriptionally inactive. However, at 32 degrees C, p53Ala143 strongly activated transcription from several physiologically relevant p53-responsive promoters, to extents similar or greater than that of wt p53. Unexpectedly, p53Ala143 was defective in inducing apoptosis in H1299 cells at 32 degrees C. Concomitantly with the loss of apoptotic activity, p53Ala143 was found to be deficient in its ability to activate transcription from the p53-responsive portions of the Bax and insulin-like growth factor-binding protein 3 gene promoters. It is proposed that there may exist distinct classes of p53-responsive promoters, whose ability to be activated by p53 can be regulated differentially. Such differential regulation may have functional consequences for the effects of p53 on cell fate.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.