Probing potential binding modes of the p53 tetramer to DNA based on the symmetries encoded in p53 response elements

Symmetries in the p53 response-element (p53RE) encode binding modes for p53 tetramer to recognize DNA. We investigated the molecular mechanisms and biological implications of the possible binding modes. The probabilities evaluated with molecular dynamics simulations and DNA sequence analyses were found to be correlated, indicating that p53 tetramer models studied here are able to read DNA sequence information. The traditionally believed mode with four p53 monomers binding at all four DNA quarter-sites does not cause linear DNA to bend. Alternatively, p53 tetramer can use only two monomers to recognize DNA sequence and induce DNA bending. With an arrangement of dimer of AB dimer observed in p53 trimer–DNA complex crystal, p53 can recognize supercoiled DNA sequence-specifically by binding to quarter-sites one and four (H14 mode) and recognize Holliday junction geometry-specifically. Examining R273H mutation and p53–DNA interactions, we found that at least three R273H monomers are needed to disable the p53 tetramer, consistent with experiments. But just one R273H monomer may greatly shift the binding mode probabilities. Our work suggests that p53 needs balanced binding modes to maintain genome stability. Inverse repeat p53REs favor the H14 mode and direct repeat p53REs may have high possibilities of other modes.     

Different recognition of DNA modified by aatitumor cisplatin and its clinically ineffective trans isomer by tumor suppressor protein p53

The p53 gene encodes a nuclear phosphoprotein that is biologically activated in response to genotoxic stresses including treatment with anticancer platinum drugs. The DNA binding activity of p53 protein is crucial for its tumor suppressor function. DNA interactions of active wild-type human p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by antitumor cisplatin and its clinically ineffective trans isomer (transplatin) were investigated by using a gel mobility shift assay. It was found that DNA adducts of cisplatin reduced binding affinity of the consensus DNA sequence to p53, whereas transplatin adducts did not. This result was interpreted to mean that the precise steric fit required for the formation and stability of the tetrameric complex of p53 with the consensus sequence cannot be attained, as a consequence of severe conformational perturbations induced in DNA by cisplatin adducts. The results also demonstrate an increase of the binding affinity of p53 to DNA lacking the consensus sequence and modified by cisplatin but not by transplatin. In addition, only major 1,2-GG intrastrand cross-links of cisplatin are responsible for this enhanced binding affinity of p53. The data base on structures of various DNA adducts of cisplatin and transplatin reveals distinctive structural features of 1,2-intrastrand cross-links of cisplatin, suggesting a unique role for this adduct in the binding of p53 to DNA lacking the consensus sequence. The results support the hypothesis that the mechanism of antitumor activity of cisplatin may also be associated with its efficiency to affect the binding affinity of platinated DNA to active p53 protein.     

Cooperative binding of tetrameric p53 to DNA

We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of approximately 3 microM for its dimer-tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.     

Efficient specific DNA binding by p53 requires both its central and C-terminal domains as revealed by studies with high-mobility group 1 protein

The nonhistone chromosomal protein high-mobility group 1 protein (HMG-1/HMGB1) can serve as an activator of p53 sequence-specific DNA binding (L. Jayaraman, N. C. Moorthy, K. G. Murthy, J. L. Manley, M. Bustin, and C. Prives, Genes Dev. 12:462-472, 1998). HMGB1 is capable of interacting with DNA in a non-sequence-specific manner and causes a significant bend in the DNA helix. Since p53 requires a significant bend in the target site, we examined whether DNA bending by HMGB1 may be involved in its enhancement of p53 sequence-specific binding. Accordingly, a 66-bp oligonucleonucleotide containing a p53 binding site was locked in a bent conformation by ligating its ends to form a microcircle. Indeed, p53 had a dramatically greater affinity for the microcircle than for the linear 66-bp DNA. Moreover, HMGB1 augmented binding to the linear DNA but not to the microcircle, suggesting that HMGB1 works by providing prebent DNA to p53. p53 contains a central core sequence-specific DNA binding region and a C-terminal region that recognizes various forms of DNA non-sequence specifically. The p53 C terminus has also been shown to serve as an autoinhibitor of core-DNA interactions. Remarkably, although the p53 C terminus inhibited p53 binding to the linear DNA, it was required for the increased affinity of p53 for the microcircle. Thus, depending on the DNA structure, the p53 C terminus can serve as a negative or a positive regulator of p53 binding to the same sequence and length of DNA. We propose that both DNA binding domains of p53 cooperate to recognize sequence and structure in genomic DNA and that HMGB1 can help to provide the optimal DNA structure for p53.     

Modulation of p53 binding to Holliday junctions and 3-cytosine bulges by phosphorylation events

Recognition of certain types of DNA lesions by the tumor suppressor protein, p53, represents one of the several downstream functions of this protein in response to DNA damage. This binding property is regulated by several factors including posttranslational modifications and interactions with other proteins. Phosphorylation by several stress-response kinases activates p53 by increasing protein stability as well as transactivation properties. Here we examined the effect of phosphorylation events on the sequence-independent binding properties of p53 using two DNA substrates: One resembling Holliday junctions and the other containing extra base bulges. Gel retardation assays showed that dephosphorylation of serine 392 in the C-terminal domain of p53 greatly reduces Holliday junction and lesion recognition. In contrast, sequence-specific binding is disrupted by the removal of some N-terminal phosphates but not serine 392. Rephosphorylation of p53 by certain kinases can restore p53 recognition of Holliday junctions and 3-cytosine bulges. In all cases, phosphorylation of serine 392 occurs; however, reactivation also involves other residues. Together, the results show that p53 DNA binding activity is strongly regulated by the phosphorylation state of the protein.     

Interaction with p53 enhances binding of cisplatin-modified DNA by high mobility group 1 protein

A nonhistone chromosomal protein, high mobility group (HMG) 1, is ubiquitous in higher eukaryotic cells and binds preferentially to cisplatin-modified DNA. HMG1 also functions as a coactivator of p53, a tumor suppressor protein. We investigated physical interactions between HMG1 and p53 and the influence of p53 on the ability of HMG1 to recognize damaged DNA. Using immunochemical coprecipitation, we observed binding of HMG1 and p53. Interaction between HMG1 and p53 required the HMG A box of HMG1 and amino acids 363-376 of p53. Cisplatin-modified DNA binding by HMG1 was significantly enhanced by p53. An HMG1-specific antibody that recognized the A box of this protein also stimulated cisplatin-modified DNA binding. These data suggest that an interaction with either p53 or antibody may induce conformational change in the HMG1 A box that optimizes DNA binding by HMG1. Interaction of p53 with HMG1 after DNA damage may promote activation of specific HMG1 binding to damaged DNA in vivo and provide a molecular link between DNA damage and p53-mediated DNA repair.     

Sequestering of p53 into DNA-protein filaments revealed by electron microscopy

Using electron microscopy, we analyzed the interaction of bacterially expressed full-length p53, p53(1-393), and its C-terminal fragment, p53(320-393), with long (approximately 3000 bp) dsDNA in linear and supercoiled (|DeltaLk| approximately 4-6) forms containing or lacking the p53 recognition sequence (p53CON). The main structural feature of the complexes formed by either protein was a DNA-protein filament, in which two DNA duplexes are linked (synapsed) via bound protein tetramers. The efficiency of the synapse, reflected in its length and the fraction of molecules exhibiting DNA-protein filaments, was significantly modulated by the molecular form of the protein and the topological state of the DNA. With linear DNA, the synapse yield promoted by the C-terminus fragment was very low, but the full-length protein was effective in linking noncontiguous duplexes, leading to the formation of intramolecular loops constrained at their bases by short regions of synapsed DNA duplexes. When the linear DNA contained p53CON, regions of preferential sequence, i.e., encompassing p53CON and probably p53CON-like sequences, were predominantly synapsed, indicating a sequence specificity of the p53 core domain. With scDNA, the synapse yield was significantly higher compared to the linear counterparts and was weakly dependent on the sign of superhelicity and presence or absence of p53CON. However, the full-length protein was more effective in promoting DNA synapses compared to the C-terminal fragment. The overall structure of the DNA-protein filaments was apparently similar for either protein form, although the apparent width differed slightly (approximately 7-9 nm and approximately 10-12 nm for p53(320-393) and p53(1-393), respectively). No distortion of the DNA helices involved in the synapse was found. We conclude that the structural similarity of DNA-protein filaments observed for both proteins is attributable mainly to the C-terminus, and that the yield is dictated by the specific and possibly nonspecific interactions of the core domain in combination with DNA topology. Possible implications for the sequestering of p53 in DNA-protein filaments are discussed.     

Differential recognition by the tumor suppressor protein p53 of DNA modified by the novel antitumor trinuclear platinum drug BBR3464 and cisplatin

The trinuclear platinum agent BBR3464, a representative of a new class of anticancer drugs, is more potent than conventional mononuclear cisplatin [cis-diamminedichloroplatinum(II)]. BBR3464 retains significant activity in human tumor cell lines and xenografts that are refractory or poorly responsive to cisplatin, and displays a high activity in human tumor cell lines that are characterized by both wild-type and mutant p53 gene. In contrast, on average, cells with mutant p53 are more resistant to the effect of cisplatin. It has been hypothesized that the sensitivity or resistance of tumor cells to cisplatin might be also associated with cell cycle control and repair processes that involve p53. DNA is a major pharmacological target of platinum compounds and DNA binding activity of the p53 protein is crucial for its tumor suppressor function. This study, using gel-mobility-shift assays, was undertaken to examine the interactions of active and latent p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by BBR3464 in a cell free medium and to compare these results with those describing the interactions of these proteins with DNA modified by cisplatin. The results indicate that structurally different DNA adducts of BBR3464 and cisplatin exhibit a different efficiency to affect the binding affinity of the modified DNA to p53 protein. It has been suggested that different structural perturbations induced in DNA by the adducts of BBR3464 and cisplatin produce a differential response to p53 protein activation and recognition and that a 'molecular approach' to control of downstream effects such as protein recognition and pathways of apoptosis induction may consist in design of structurally unique DNA adducts as cell signals.     

p53 Binding to Nucleosomal DNA Depends on the Rotational Positioning of DNA Response Element

The sequence-specific binding to DNA is crucial for the p53 tumor suppressor function. To investigate the constraints imposed on p53-DNA recognition by nucleosomal organization, we studied binding of the p53 DNA binding domain (p53DBD) and full-length wild-type p53 protein to a single p53 response element (p53RE) placed near the nucleosomal dyad in six rotational settings. We demonstrate that the strongest p53 binding occurs when the p53RE in the nucleosome is bent in the same direction as observed for the p53-DNA complexes in solution and in co-crystals. The p53RE becomes inaccessible, however, if its orientation in the core particle is changed by ∼180°. Our observations indicate that the orientation of the binding sites on a nucleosome may play a significant role in the initial p53-DNA recognition and subsequent cofactor recruitment.     

Effect of p53 protein redox states on binding to supercoiled and linear DNA.

The binding of p53 to its DNA consensus sequence is modulated by the redox state of the protein in vitro. We have shown previously that reduced wild-type p53 binds strongly to supercoiled DNA (scDNA) regardless of the presence or absence of p53CON. Here we compare the effects of oxidation of p53 by azodicarboxylic acid bis[dimethylamide] (diamide) and other agents on p53 binding to p53CON and to scDNA. Oxidation decreases the binding of p53 to scDNA; however, under conditions where binding to p53CON in a DNA fragment is completely abolished, some residual binding to scDNA is still observed. Increasing the concentration of oxidized p53 confers minimal changes in p53 binding to both scDNA and p53CON. Reduction of the oxidized protein by dithiothreitol neither restores its binding to DNA nor to p53CON in DNA fragments. In the presence of excess zinc ions, oxidation of p53 is, however, reversible. We conclude that the irreversibility of p53 oxidation is due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain accompanied by a conformational change of the p53 molecule after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein.     

p53 binds to cisplatin-damaged DNA

We have previously shown that bacterially expressed p53 protein or p53 protein isolated from cis-diamminedichloroplatinum II (cisplatin)-damaged cells is capable of binding to double-stranded platinated DNA molecules lacking any p53 DNA binding sites. Here we report using various p53 mutants that two separate domains of p53 protein affect p53 binding to platinated DNA. Mutations within the central core of p53, the domain responsible for sequence-specific DNA binding activity, completely eliminated p53 binding to platinated DNA. Based on competition experiments p53 preferred binding to sequence-specific DNA molecules over platinated DNA molecules. However, p53 binding to platinated DNA molecules was significantly stronger than p53 interactions with DNA molecules lacking damage and a p53 consensus site. Finally, an antibody specific to the C-terminal domain of p53 (pAb421) which activates sequence-specific DNA binding activity inhibited p53 binding to platinated DNA. Taken together, these results suggest that in addition to binding to p53 DNA binding sites, p53 also interacts with cisplatin-damaged DNA molecules.     

Recognition of DNA modified by trans-[PtClNH(4-hydroxymethylpyridine)] by tumor suppressor protein p53 and character of DNA adducts of this cytotoxic complex

trans-[PtCl(2)NH(3)(4-Hydroxymethylpyridine)] (trans-PtHMP) is an analogue of clinically ineffective transplatin, which is cytotoxic in the human leukemia cancer cell line. As DNA is a major pharmacological target of antitumor platinum compounds, modifications of DNA by trans-PtHMP and recognition of these modifications by active tumor suppressor protein p53 were studied in cell-free media using the methods of molecular biology and biophysics. Our results demonstrate that the replacement of the NH(3) group in transplatin by the 4-hydroxymethylpyridine ligand affects the character of DNA adducts of parent transplatin. The binding of trans-PtHMP is slower, although equally sequence-specific. This platinum complex also forms on double-stranded DNA stable intrastrand and interstrand cross-links, which distort DNA conformation in a unique way. The most pronounced conformational alterations are associated with a local DNA unwinding, which was considerably higher than those produced by other bifunctional platinum compounds. DNA adducts of trans-PtHMP also reduce the affinity of the p53 protein to its consensus DNA sequence. Thus, downstream effects modulated by recognition and binding of p53 protein to DNA distorted by trans-PtHMP and transplatin are not likely to be the same. It has been suggested that these different effects may contribute to different antitumor effects of these two transplatinum compounds.     

p53 C-terminal interaction with DNA ends and gaps has opposing effect on specific DNA binding by the core

In addition to binding DNA in a sequence-specific manner, the p53 tumour suppressor protein can interact with damaged DNA. In order to understand which structural features in DNA the C-teminal domain recognises we have studied the interaction of p53 protein with different types of DNA oligonucleotides imitating damaged DNA. Here we show that one unpaired nucleotide within double-stranded (ds)DNA is sufficient for recognition by the p53 C-terminus, either as a protruding end or as an internal gap in dsDNA. C-terminal interaction with DNA ends facilitated core domain binding to DNA, whereas interaction with gaps prevented core domain–DNA complexing, implying that p53 might adopt distinct conformations upon binding to different DNA lesions. These observations suggest that both single-strand and double-strand breaks can serve as a target for p53 C-terminal recognition in vivo and indicate that p53 might recruit different repair factors to the sites of damaged DNA depending on the type of lesion.