Stimulation of polyomavirus DNA replication by wild-type p53 through the DNA-binding site.

The tumor suppressor p53 possesses characteristics of a transcription factor; it binds to specific DNA sequences and activates transcription from various promoters. Here we found that murine wild-type p53 stimulated not only transcription but also polyomavirus (Py) DNA replication in a sequence-dependent manner. Oncogenic mutant p53, lacking the DNA-binding activity, showed no stimulation of Py DNA replication. Deletion of the N-terminal acidic transactivation domain of wild-type p53, which completely eliminated the ability to stimulate transcription, only impaired the function to stimulate Py DNA replication. The replication-stimulating activity of wild-type p53 was impaired by the deletion of the C-terminal oligomerization domain as well, without affecting the ability to stimulate transcription. The region responsible for the sequence-specific DNA-binding activity mapped to the central portion of the p53 molecule has a minimal activity. The results indicate that both the N-terminal and the C-terminal regions significantly contribute to the p53-mediated stimulation of Py DNA replication.

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CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

Journal of Biological Chemistry ◽

10.1074/jbc.m401854200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45887-45896 ◽

Cited By ~ 45

Author(s):

Mark J. Demma ◽

Serena Wong ◽

Eugene Maxwell ◽

Bimalendu Dasmahapatra

Keyword(s):

Dna Binding ◽

Mammalian Cells ◽

P53 Protein ◽

Binding Activity ◽

Mutant P53 ◽

Dependent Manner ◽

Wild Type ◽

Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

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Stimulation of p53 DNA Binding by c-Abl Requires the p53 C Terminus and Tetramerization

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.741-748.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 741-748 ◽

Cited By ~ 46

Author(s):

Ying Nie ◽

Heng-Hong Li ◽

Craig M. Bula ◽

Xuan Liu

Keyword(s):

Dna Binding ◽

Binding Activity ◽

Carboxyl Terminus ◽

Dependent Manner ◽

Regulatory Domain ◽

Dna Binding Activity ◽

C Terminus ◽

Carboxyl Terminal ◽

Dna Complex ◽

Stimulation Of

ABSTRACT The carboxyl terminus of p53 is a target of a variety of signals for regulation of p53 DNA binding. Growth suppressor c-Abl interacts with p53 in response to DNA damage and overexpression of c-Abl leads to G1 growth arrest in a p53-dependent manner. Here, we show that c-Abl binds directly to the carboxyl-terminal regulatory domain of p53 and that this interaction requires tetramerization of p53. Importantly, we demonstrate that c-Abl stimulates the DNA-binding activity of wild-type p53 but not of a carboxyl-terminally truncated p53 (p53Δ363C). A deletion mutant of c-Abl that does not bind to p53 is also incapable of activating p53 DNA binding. These data suggest that the binding to the p53 carboxyl terminus is necessary for c-Abl stimulation. To investigate the mechanism for this activation, we have also shown that c-Abl stabilizes the p53-DNA complex. These results led us to hypothesize that the interaction of c-Abl with the C terminus of p53 may stabilize the p53 tetrameric conformation, resulting in a more stable p53-DNA complex. Interestingly, the stimulation of p53 DNA-binding by c-Abl does not require its tyrosine kinase activity, indicating a kinase-independent function for c-Abl. Together, these results suggest a detailed mechanism by which c-Abl activates p53 DNA-binding via the carboxyl-terminal regulatory domain and tetramerization.

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DNA-Binding Activity of Wild-Type p53 Protein is Mediated by the Central Part of the Molecule and Controlled by Its C Terminus

Cancer Detection and Prevention ◽

10.1046/j.1525-1500.1998.00003.x ◽

1998 ◽

Vol 22 (1) ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Roland Wolkowicz ◽

Amnon Peled ◽

Barry Elkind ◽

Varda Rotter

Keyword(s):

Dna Binding ◽

P53 Protein ◽

Binding Activity ◽

Wild Type ◽

Dna Binding Activity ◽

C Terminus ◽

Wild Type P53

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Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5699 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5699-5706 ◽

Cited By ~ 120

Author(s):

G W Verhaegh ◽

M J Richard ◽

P Hainaut

Keyword(s):

Dna Binding ◽

Metal Ions ◽

Copper Ions ◽

P53 Protein ◽

Binding Activity ◽

Redox Activity ◽

Wild Type ◽

Dna Binding Activity ◽

Wild Type P53

Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.

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Mutants of the tumour suppressor p53 L1 loop as second-site suppressors for restoring DNA binding to oncogenic p53 mutations: structural and biochemical insights

Biochemical Journal ◽

10.1042/bj20091888 ◽

2010 ◽

Vol 427 (2) ◽

pp. 225-236 ◽

Cited By ~ 25

Author(s):

Assia Merabet ◽

Hellen Houlleberghs ◽

Kate Maclagan ◽

Ester Akanho ◽

Tam T. T. Bui ◽

...

Keyword(s):

Dna Binding ◽

Double Mutant ◽

Hot Spot ◽

Binding Activity ◽

Tumour Suppressor ◽

Wild Type ◽

Dna Binding Activity ◽

Wild Type P53 ◽

Dynamics Simulations ◽

Tumour Suppressor P53

To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 ‘hot spot’ mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53; (iv) the effect of H115N as a second-site suppressor to restore DNA-binding activity is specific to R248Q, but not to R248W; (v) molecular-dynamics simulations indicate that R248Q/H115N has a conformation similar to wild-type p53, which is distinct from that of R248Q. These findings could be exploited in designing strategies for cancer therapy to identify molecules that could mimic the effect of H115N in restoring function to oncogenic p53 mutants.

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Structure of the p53/RNA polymerase II assembly

Communications Biology ◽

10.1038/s42003-021-01934-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Shu-Hao Liou ◽

Sameer K. Singh ◽

Robert H. Singer ◽

Robert A. Coleman ◽

Wei-Li Liu

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Conformational Changes ◽

P53 Protein ◽

Binding Activity ◽

Transactivation Domain ◽

Pol Ii ◽

Dna Binding Activity ◽

Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

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DNA Damage Induced Hyperphosphorylation of Replication Protein A. 2. Characterization of DNA Binding Activity, Protein Interactions, and Activity in DNA Replication and Repair†

Biochemistry ◽

10.1021/bi048057b ◽

2005 ◽

Vol 44 (23) ◽

pp. 8438-8448 ◽

Cited By ~ 34

Author(s):

Steve M. Patrick ◽

Greg G. Oakley ◽

Kathleen Dixon ◽

John J. Turchi

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Binding ◽

Protein Interactions ◽

Protein A ◽

Replication Protein A ◽

Binding Activity ◽

Dna Binding Activity ◽

Dna Replication And Repair

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Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2464-2476.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2464-2476

Author(s):

M Cockell ◽

B J Stevenson ◽

M Strubin ◽

O Hagenbüchle ◽

P K Wellauer

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Dna Interaction ◽

Binding Activity ◽

Alpha Amylase ◽

Dna Binding Activity ◽

A Cell ◽

Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

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Transformation by Fos proteins requires a C-terminal transactivation domain

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7429-7438.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7429-7438

Author(s):

R Wisdon ◽

I M Verma

Keyword(s):

Dna Binding ◽

Leucine Zipper ◽

Fibroblast Cell ◽

Binding Activity ◽

Sequence Specificity ◽

Transactivation Domain ◽

Functional Requirements ◽

Dna Binding Activity ◽

The Difference ◽

Transforming Proteins

The Fos family of proteins now includes seven members: the retroviral proteins FBR-v-Fos and FBJ-v-Fos and the cellular proteins c-Fos, FosB, FosB2, Fra1, and Fra2. Four proteins (FBR-v-Fos, FBJ-v-Fos, c-Fos, and FosB) transform established rodent fibroblast cell lines, while three (FosB2, Fra1, and Fra2) do not. As all family members display sequence-specific DNA-binding activity as part of a heterodimeric complex with Jun proteins, other features must account for the differences in transforming potential. We demonstrate here that all transforming members have a C-terminal transactivation domain that is lacking in nontransforming members. The nontransforming proteins Fra1 and Fra2 can be converted to transforming proteins by fusion of a transactivation domain from either FosB or VP16. We also demonstrate that differences in the basic region-leucine zipper domain affecting either the affinity or sequence specificity of DNA binding are not determinants of the difference in transforming potential among members of the Fos family. The results further define the functional requirements for transformation by Fos proteins and suggest that the subunit composition of AP1 complexes is an important determinant of mitogenic signalling capability.

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