Abstract

We showed previously that bacterially expressed full‐length human wild‐type p53b(ane–393) binds selectively to supercoiled (sc)Dna in sc/linear Deoxyribonucleic acid competition experiments, a process nosotros termed supercoil‐selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C‐terminal domain (amino acids 347–382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361–393) poly peptide (defective the p53 tetramerization domain, amino acids 325–356) did not exhibit SCS binding while both dimeric mutant p53(319– 393)L344A and fusion protein GCN4–p53(347–393) were constructive in SCS binding. Supershifting of p53(320–393)–scDNA complexes with monoclonal antibodies revealed that the amino acid region 375–378, constituting the epitope of the Bp53‐10.1 antibiotic, plays a role in binding of the p53(320–393) protein to scDNA. Using electron microscopy nosotros observed p53–scDNA nucleoprotein filaments produced by all the C‐final proteins that displayed SCS binding in the gel electrophoresis experiments; no filaments formed with the monomeric p53(361– 393) protein. We propose a model co-ordinate to which two DNA duplexes are compacted into p53–scDNA filaments and talk over a function for filament formation in recombination.

Received May 23, 2002; Revised August 27, 2002; Accepted September 20, 2002

INTRODUCTION

The p53 tumor suppressor poly peptide protects cells from malignant transformation by regulating the responses of cell growth and death to genotoxic agents (reviewed in i – 3 ). Stress‐induced agile p53 poly peptide triggers growth arrest or cell expiry by apoptosis at to the lowest degree in part via transcriptional activation of a gear up of genes containing p53 recognition sites. In an unstressed prison cell, p53 participates in various processes of Deoxyribonucleic acid repair and DNA recombination by virtue of its ability to interact with protein components of the repair and recombination machineries and via various DNA‐binding activities ( 4 ).

p53 protein contains 393 amino acids that contain several functional domains ( v , 6 ). The Northward‐final domain (amino acids 1– to ∼100) encompasses a transactivation region (amino acids 1–42) and a proline‐rich region with v copies of the sequence PXXP (amino acids 61–94). The evolutionarily highly conserved key (core) domain (amino acids ∼100 to ∼300) is involved in sequence‐specific bounden to promoters of p53‐regulated genes ( 7 ). This domain also interacts with internal regions of unmarried‐stranded (ss)DNA ( viii ), three‐stranded Dna substrates mimicking early recombination intermediates ( 9 ), insertion/deletion mismatches ( 10 ) and cruciforms ( xi ) and likewise manifests a 3′→v′ exonuclease activity ( 12 ). Point mutations in this domain are the most frequent alterations in p53 found in human cancers ( 13 ). The C‐terminal region of the protein contains a flexible linker (amino acids ∼300 to ∼325), a tetramerization domain (amino acids ∼325–356) and a bones C‐terminal Deoxyribonucleic acid‐binding domain (amino acids 363–382). The ability of the C‐terminus to bind to single‐stranded gaps in double‐stranded (ds)DNA ( 14 ), γ‐irradiated DNA in vitro ( xv ) and ssDNA ends and to catalyze DNA strand transfer ( 8 , 16 ) presumably accounts for the office of p53 in DNA repair and recombination.

Recently, it was shown that p53 protein binds strongly to negatively as well every bit to positively supercoiled (sc) plasmid DNA ( 17 , eighteen ). It has been suggested that both the cadre domain and the C‐concluding domain regulate the bounden of p53 to scDNA ( 17 – 19 ). Competition between scDNAs and their linearized (lin) forms revealed a strong preference for scDNAs by wild‐type p53, suggesting a new p53–DNA binding mode denoted supercoil‐selective (SCS) binding ( twenty ). The p53 core domain exhibited only weak preferential binding to scDNA in the sc/lin competition assay, supporting the previous notion that some other domain (probably C‐terminal) is involved in selective binding of the full‐length p53 to scDNA ( 20 , 21 ).

Several proteins bind preferentially to scDNA. For example, prokaryotic topoisomerase I (ω protein) distinguishes DNA topology by binding to local regions with single‐stranded character stabilized past the underwinding of the double helix ( 22 ). It has been proposed that the preference of eukaryotic topoisomerases and HMG box‐containing proteins (e.g. chromosomal HMG1 and xUBF transcription factor) for crossovers in scDNA accounts for their ability to distinguish the topological land of both negatively and positively scDNA ( 23 , 24 ).

In this report, we accept analyzed the contributions of the p53 domains to preferential binding to scDNA. Using bacterially expressed p53 deletion mutants and chimeric proteins we demonstrate that dimerization of the C‐concluding segment is a prerequisite for the stiff SCS bounden of p53. Visualization of p53–scDNA complexes by electron microscopy reveal the ability of p53 dimers to stabilize two DNA duplexes in close vicinity, leading ultimately to the formation of nucleoprotein filaments.

MATERIALS AND METHODS

Recombinant plasmids

Plasmids encoding human wild‐blazon p53(1–393), p53(i–363), p53(45–349) and p53(44–393) were kindly provided by C. Midgley ( 25 ). Core domain‐containing recombinant plasmid p53(94–312) was from S. Gorina ( 26 ). C‐terminal p53 fragment p53(320–393) inserted in pGEX‐2T vector was from Yard. Protopopova ( 8 , fourteen ). Figure i A schematically shows the p53 fragments expressed from these plasmids.

PCR and standard cloning procedures were used ( 27 ) to set plasmids for expression of p53 C‐last proteins. To clone p53(319–393)L344A mutant, p53 cDNA (kindly provided by M. Thou. Luciani) containing a substitution of CTG by GCG (Leu→Ala at amino acid position 344) was used as a template for PCR amplification of the p53 region coding for amino acid residues 319–393. The dimerization domain (amino acids 252–280) of transcription cistron GCN4 (plasmid pLZ335 kindly provided by T. Halazonetis; 28 ) was PCR amplified and inserted into plasmid pGEX containing p53(347–393) to create chimeric GCN4–p53(347–393) protein. The p53 insert in plasmid pGEX‐p53(363–393) started from the AGG codon for K363; the artificial G361 and S362 codons originated from the pGEX vector. Since thrombin cleavage (see below) leaves both amino acids Chiliad and S fused to p53 protein, the final production contained amino acrid residues identical to p53(361–393). Deoxyribonucleic acid sequencing of each plasmid confirmed that no additional mutations were introduced into the p53 coding regions during PCR.

Supercoiled pBluescript SK Ii plasmid DNA ( 17 ) was isolated from bacterial strain TOP10 every bit described in the Qiagen protocol (Qiagen, Deutschland). Sma I brake enzyme (Takara, Japan) was used for linearization of pBluescript SK II (Stratagene).

p53 recombinant proteins purification

Each of the p53 constructs was expressed in Escherichia coli strain BL21 using a ii‐step induction at 18°C to limit protein aggregation ( 29 ). The proteins p53b(1–393), p53(one–363), p53(44–393) and p53(45–349) were purified co-ordinate to a protocol described previously ( 30 ) with some modifications. Bacterial lysate was loaded at 4°C and 1 ml/min onto a 5 ml HiTrap–heparin column (Pharmacia, Sweden) that had been pre‐equilibrated in buffer A (10% glycerol, 25 mM Na HEPES, pH 7.6, 5 mM DTT, 1 mM benzamidine) with fifty mM KCl. After washing the column with 8 cavalcade vol of buffer A containing fifty mM KCl, an 8 column vol linear slope (from 0.05 to i M KCl in buffer A) was initiated at 0.5 ml/min. The p53 forms eluted at 0.v M KCl [p53b(one–393)] or 0.4 M KCl [p53(1–363), p53(44–393) and p53(45–349)]. p53‐containing fractions were concentrated using ammonium sulfate precipitation and MICROSEP 10K centrifugation (Drape Filtron, Germany). Samples were then applied to a Superdex 200 cavalcade (HR 10/30; Pharmacia, Sweden) equilibrated in buffer A with 200 mM KCl. The peak fractions of p53b(1–393), p53(1–363) and/or p53(44–393) were eluted after 15, 22 and/or 27 min (flow charge per unit 0.five ml/min), respectively. p53(94–312) was isolated as described previously ( 29 ).

Proteins p53(320–393), p53(319–393)L344A, p53(361–393) and GCN4–p53(347–393) were expressed every bit fusions with GST. Lysis of bacteria was performed in 1× PBS (150 mM NaCl, 3 mM KCl, x mM Na 2 HPO iv , 2 mM NaH 2 PO 4 , pH 7.three), 0.1% Triton X‐100, 1 mM benzamidine and 0.1 mM PMSF. The lysate was loaded onto a v ml HiTrap‐GST cavalcade (Pharmacia, Sweden). Afterward washing with ane× PBS (10 column vol), the fusion protein was eluted with x–20 mM reduced glutathione. Protein‐containing fractions were loaded onto MonoS columns (Hour five/5; Pharmacia, Sweden) and eluted with a 40 ml linear gradient (from 0 to 1 M KCl in buffer A). The GST tag was cleaved with 1 U thrombin (Calbiochem, Usa) per 5 mg fusion protein. Except for the p53(361–393) protein (separated on MICROSEP 10K), each p53 poly peptide was separated from GST protein on MonoS columns using a 40 ml linear slope (0–ane Chiliad NaCl in l mM Tris–HCl, pH eight.8, or 25 mM Na HEPES, pH 7.half-dozen). Most of the pure p53 proteins eluted at 0.five M NaCl ( 31 ). The purity and appropriate size of each protein was analyzed with Coomassie blue staining of SDS–Page gels (Fig. 1 B) and western blotting (not shown). Mouse monoclonal antibodies (mAb) Practise‐one (recognizing amino acids 20–25), Exercise‐11 (amino acids 176–185), Do‐12 (amino acids 256–270), Bp53‐6.1 (amino acids 381–390), Bp53‐ten.ane (amino acids 375–379) and ICA‐9 (amino acids 388–393) were applied to detect epitopes present within the p53 fragments.

Competition assay

Contest experiments with a mix of supercoiled (native superhelix density) and Sma I linearized pBluescript SK II DNAs (in equimolar concentrations) were performed as described previously ( 20 ). The total amount of DNA was in the range 0.three–1.six µg (higher amounts of DNA and p53 were used in samples with low p53/Dna ratios to keep the p53 level detectable for monoclonal antibodies). The lin/scDNA mix was incubated with p53 fragment (p53 tetramer/Dna molar ratio = 1–twenty) in binding buffer (v mM Tris–HCl, pH 7.6, 0.5 mM EDTA, 50 mM KCl and 0.01% Triton X‐100) for 30 min at 0°C. Samples were loaded onto a 1.3% agarose gel containing 0.33× Tris–borate–EDTA (TBE) buffer; in this arrangement linDNA migrates faster than scDNA. Afterward 8–12 h electrophoresis (at 4–half dozen V/cm), gels were blotted onto a nitrocellulose transfer membrane Protran R (Schleicher and Schuell, Germany) and each p53 fragment was detected with the corresponding mAb using the ECL detection system ( 20 ).

Supershifting experiment

p53(320–393) protein was incubated with scDNA (0.iii µg) for 15 min on ice (p53 tetramer/Deoxyribonucleic acid molar ratio = v). Then, affinity purified mouse mAb was added (mAb/p53 tetramer molar ratio = 1.5 or 3) and incubation connected for a further fifteen min at room temperature ( 32 ). Samples were run on a 1% agarose gel in 0.33× TBE and stained with ethidium bromide. The gel was blotted and p53 protein detected using rabbit anti‐p53 polyclonal CM1 antibody.

Electron microscopy

Complexes of p53 with Dna were formed in a buffer containing ten mM Na HEPES, pH 7.5, 50 mM KCl, 0.01% Triton X‐100. p53 constructs (final concentration 100 or 500 nM where indicated for a given stable oligomeric land in solution) were added to a reaction mixture containing equimolar concentrations of linear and native supercoiled pBluescript DNA (5 nM each). Samples were incubated on ice for xxx–60 min and an aliquot was withdrawn and diluted 20‐fold into 10 mM Na HEPES, pH 7.5, 10 mM KCl. Electron microscopic samples were prepared every bit described ( 29 ). Samples were analyzed with a Philips CM12 electron microscope operated in a tilted night field manner. Images were scanned using Agfa DuoScan T2500 (Agfa, Frg). For printing, images were flattened with a loftier‐laissez passer filter (radius 250 pixels) and subsequently adapted for contrast/effulgence using Adobe Photoshop.

RESULTS

The C‐terminal segment of p53 is important for p53 SCS binding

We studied the interaction of bacterially expressed truncated forms of human being p53 (Fig. 1 ) with linDNA and scDNA in order to elucidate the relative contributions of unlike domains to the preferential binding of p53 to scDNA ( 17 , 20 ). An equimolar mixture of supercoiled and linear ( Sma I linearized) forms of pBluescript DNA was incubated with various p53 fragments and the formation of both p53–scDNA and p53–linDNA complexes was analyzed by agarose gel electrophoresis (Fig. 2 A, left) and immunoblotting assay of the agarose gel (Fig. 2 A, right, and B) as described ( 20 ). Notation that under the conditions used (ane.three% agarose, 0.33× TBE buffer) linDNA migrates faster than scDNA (Fig. ii A, compare lanes 1 and 3). Incubation of either scDNA (lane 2) or linDNA (lane 4) with p53b(1–393) protein led to the appearance of a new band(s) located to a higher place the position of the corresponding free DNA. Superposition of the left and right panels conspicuously shows that p53 was located exclusively inside retarded bands, indicating that these bands represent p53–Dna complexes. When the equimolar mixture of sc/linDNA was incubated with an increasing corporeality of p53b(ane–393) protein (at either 0 or 37°C) only p53–scDNA complexes were observed (Fig. 2 , lanes half-dozen and 9–eleven; information for 37°C not shown).

Information technology is evident that p53b(1–393) displays very high selectivity for scDNA in a sc/linDNA competition assay (Fig. ii A, lanes vi and 9–11, and B, lanes 1 and 2). This selective bounden of p53 to scDNA was termed SCS binding ( 20 ). The selectivity for scDNA binding was due to topological constraints not dependent on the presence of Deoxyribonucleic acid ends, since p53 bound similarly to relaxed round DNA as to linear templates (data not shown). In dissimilarity, p53(1–363), defective the basic C‐concluding DNA‐bounden domain (amino acids 363–382), formed both p53–scDNA and p53–linDNA complexes (Fig. ii B, lanes 3 and 4) with a weak preference for scDNA. Similarly, both the p53(45–349) and p53(94–312) proteins, containing the p53 cadre domain, showed only a weak preference for scDNA (Fig. 2 B, lanes 5–8). Taken together, these data imply an essential role of the p53 C‐final region in SCS binding.

To further analyze the function of the p53 C‐concluding region we purified two Due north‐terminally truncated deletion mutants p53(44–393) and p53(320–393). In the sc/lin competition experiments, both proteins leap scDNA with high selectivity (Fig. ii B, lanes nine–12), suggesting that the last ∼70 amino acid residues (amino acids 320–393) of the C‐terminal role are sufficient for SCS binding.

Amino acid residues 375–378 of the basic C‐terminal region (amino acids 363–382) participate in binding to scDNA

We mapped the Dna‐bounden site (Fig. 3 A) of the C‐terminal poly peptide p53(320–393) using specific mAbs. scDNA (Fig iii B, lane one) was incubated with the p53(320–393) protein forming a p53(320–393)–scDNA complex that migrated with slightly lower mobility than free scDNA in agarose gels (Fig. 3 B, lane 2). After addition of antibody ICA‐9 (recognizing amino acids 388–393) ( 33 ) the p53(320–393)–scDNA complex was retarded further (supershifted), suggesting the formation of large (ICA‐ix)–p53(320–393)–scDNA complexes (Fig. 3 B, lanes 7 and 8). The Bp53‐six.1 epitope (amino acids 381–390) ( 34 ) in the p53(320–393)–scDNA circuitous was also accessible to the mAb, as indicated by smearing of the ring (Fig. three B, lanes 5 and 6). In agreement with these results, the p53(319–382) protein (defective the final 11 amino acrid residues of the C‐terminus) selectively bound to scDNA (data not shown), suggesting that the extreme C‐final region is not essential for SCS binding.

Addition of mouse antibody Bp53‐10.ane (recognizing amino acids 375–378) ( 34 ) to the p53(320–393)–scDNA complex did not result in supershifting (Fig. 3 B, lanes 2–4). To check p53 binding to scDNA the gel was blotted and analyzed for p53 protein using the rabbit antibody CM1 (Fig. 3 C). The p53(320–393) poly peptide was dissociated from the scDNA (lanes 3 and 4) and fully titrated with Bp53‐10.1 antibodies (lane iv). These results suggest that the Bp53‐x.1 antibody competed with scDNA for binding to identical p53 amino acrid residues. Similar results have been obtained with antibiotic pAb421 recognizing amino acids 371–380 (data non shown). We infer that amino acid residues 375–378 participate in bounden of the p53(320–393) poly peptide to scDNA.

The oligomeric land of the p53 C‐final segment is critical for its SCS binding

To analyze the role of the tetramerization domain (amino acids 325–356) we used the C‐last protein p53(319–393) L344A (containing an Ala commutation at amino acid residuum L344) and the GCN4–p53(347–393) fusion protein (containing the dimerization domain of yeast transcription factor GCN4 fused to the p53 C‐final segment). The composition of both proteins express their oligomeric state in solution to dimers (information not shown) ( 28 , 35 ). In the sc/lin competition assay, mutant p53(319–393)L344A as well as the GCN4– p53(347–393) fusion poly peptide selectively spring scDNA (Fig. 4 , lanes 4–6 and 7–9, respectively), showing for the first time that not only tetramers simply as well p53 dimers brandish SCS binding and that the p53 tetramerization domain is replaceable past a dimerization domain. In agreement with this result, glutathione S ‐transferase (GST)–p53(347–393) fusion protein (GST is a homodimer in solution; 36 ) bound selectively to scDNA (data not shown). On the other paw, neither p53(361–393) nor p53(347–393) monomeric proteins showed any preference for scDNA, forming poly peptide–Deoxyribonucleic acid complexes with both linDNA and scDNA in the sc/lin contest assay (Fig. 4 , lanes x–12 and data not shown). These results imply that the oligomeric country is disquisitional for SCS binding.

Strong SCS binding of p53 C‐terminal proteins correlates with their ability to form DNA–protein filaments

We employed electron microscopy to analyze p53 selective binding to scDNA in the equimolar mixture of scDNA and linDNA (5 nM each). For better discrimination between free and protein‐leap scDNA, the samples were deposited from 10 mM KCl since under these weather scDNA does not display a clear plectonemic grade just rather a partially open or irregular configuration (Fig. 5 E) ( 37 ). The p53(320–393) protein (100 nM, i.e. protein/DNA molar ratio = 10) showed a strong selective binding to scDNA, forming long Deoxyribonucleic acid– protein filaments, whereas practically no interaction was detected with linDNA (Fig. 5 A). Both p53(319–393) L344A and GCN4–p53(347–393), which are dimers in solution ( 28 , 35 ), also exhibited selective binding to scDNA, with complexes resembling those formed by p53(320–393), although with minor differences (Fig. 5 B and C). At poly peptide/Dna molar ratio = 10, both dimeric proteins formed complexes with less clear filamentous structure (Fig. 5 B, upper inset, and C). The regular filamentous structures were observed at higher protein/Deoxyribonucleic acid molar ratios (Fig. five B, lower inset, and C, inset). In contrast, p53(361–393) neither generated filaments with diverse Deoxyribonucleic acid targets nor did it perturb the scDNA configuration (Fig. 5 D). Other possible complexes were not detected, probably due to their low molecular mass. In view of the above data, we infer that at that place is a strong correlation between the ability of p53 proteins to selectively demark scDNA in the sc/linDNA competition experiments (Fig. four ) and to form Dna–protein filaments (Fig. 5 ).

DISCUSSION

Role of p53 domains in SCS bounden

In this work nosotros accept analyzed the preferential binding of dissimilar p53 domains to scDNA. Our previous results suggested that both the core domain and the C‐terminal function of p53 participate in the preferential binding of total‐length p53 to scDNA ( 17 , 19 , 38 ). Recently, we showed that the isolated core domain displays a preference for binding scDNA in sc/lin competition experiments, although this bias is much weaker than that observed with the bacterially expressed total‐length p53b(1–393) ( 20 ). Hither we demonstrate that the C‐last region (amino acids 320–393) exhibits SCS bounden to scDNA comparable to that of the p53b(one–393) poly peptide (Fig. 2 ), suggesting that participation of the C‐terminal region is of critical importance for scDNA selectivity. Considering the role of the core domain in SCS binding by p53b(1–393), it is possible that either the core domain modulates the C‐terminus SCS bounden in p53b(one–393) or that the mechanism of p53b(1–393) binding to scDNA differs fundamentally from that of the C‐terminal segment, i.eastward. involving direct core domain interactions likewise. The hypothesis that other domains may modulate bounden of the C‐terminal segment is supported by our ascertainment that fusion of a GST tag to the p53(320–393) protein diminishes bounden to scDNA (M.Brázdová, J.Paleček and D.I.Cherny, unpublished results).

Neither the p53(361–393) nor p53(347–393) poly peptide bound selectively to scDNA (Figs 4 and v ). We conclude from these findings that the p53 tetramerization domain (amino acids 325–356), responsible for the oligomerization state of the poly peptide, is disquisitional for SCS binding past the p53(320–393) protein. This decision is in understanding with Mazur et al . ( 18 ), who demonstrated that monomeric p53(319–393)AAA protein (containing Ala substitutions at positions L323, Y327 and L330) did not showroom preferential binding to scDNA. Nosotros have shown that the p53 C‐terminal segment (amino acids 347–393) acquires the ability to selectively bind to scDNA upon fusion to the yeast GCN4 dimerization domain (Figs 4 and 5 ). These results suggest that (i) the p53 tetramerization domain is replaceable with a dimerization domain (east.g. GCN4) and (2) the oligomeric state of the p53 C‐final segment plays a dominant function in SCS binding relative to amino acid composition of the tetramerization domain (amino acids 325–356). Withal, the amino acid segment 347–356 of this region appears necessary for SCS binding since the GCN4–p53(356–393) fusion protein showed no selectivity for scDNA in the sc/lin competition assay (J.Paleček, unpublished results) while the GCN4–p53(347–393) construct exhibited SCS bounden (Fig. 4 ). Thus, amino acrid residues 347–356 contribute either to a proper conformation of the GCN4–p53(347–393) fusion protein or participate directly in the p53–scDNA interactions.

From the successful functional replacement of the p53 tetramerization domain with the GCN4 dimerization domain, it appears that dimers of the C‐last segment (amino acids 347–393) are sufficient for SCS bounden. Thus, dimerization modulates the DNA bounden selectivity of the p53 C‐terminal region (see below; Fig. 6 ).

p53 dimers demark two dsDNA duplexes forming filamentous structures

Our recent studies suggested several potential p53‐binding motifs within scDNA. It was proposed ( xx ) that the weak preference of p53(94–312) for scDNA may reverberate binding to the internal single‐stranded regions ( 8 ), maybe occurring in native negatively scDNA only absent in linDNA ( 39 ). Jett et al. ( eleven ) reported binding of the p53(94–312) poly peptide to a cruciform arising in scDNA containing an (AT) 34 insert. Using electron microscopy we found only a small fraction of globular complexes formed by p53(94–312), p53(i–363) and insect cell‐expressed total‐length p53i(one–393) at scDNA crossovers (D.I.Cherny, M.Brázdová, J.Paleček, East.Paleček and T.Thousand.Jovin, submitted for publication). From these data we hypothesize that the interaction of p53(94–312) core domain (and the other proteins defective the C‐terminus and/or containing inactive C‐terminus) with any of the higher up motifs may contribute to the weak preferential bounden to scDNA.

The power of the p53 C‐concluding proteins to course filaments correlates well with their strong selective binding to scDNA observed in gel shift sc/lin competition experiments (Figs 4 and v ). Analysis of the p53–scDNA filaments showed that 2 DNA segments from opposing (distant) duplex strands are linked via p53 proteins (Figs five and vi ). The loftier probability of two Dna duplexes coming together in scDNA could account for the power of p53 to bind scDNA with high selectivity in the sc/lin contest experiments ( 37 ). The most attractive explanation for SCS bounden of the C‐terminal proteins is that the protein dimers (and tetramers) bind, well-nigh probably in a cooperative mode, DNA regions with shut lateral contacts inherent to the plectonemic form of natural negatively scDNA. In this regard it is worth noting that filaments formed with scDNA containing only a modest number of supercoils, e.one thousand. about five or half-dozen (D.I.Cherny, Thousand.Brázdová, J.Paleček, E.Paleček and T.One thousand.Jovin, submitted for publication).

All incubations of the p53 proteins with DNA were carried out under weather (50 mM KCl) in which scDNA has the plectonemic form ( 37 , 40 ). The conditions for electron microscopic sample grooming (10 mM KCl) were chosen such that scDNA adopted a loosely interwound configuration of irregular shape (Fig. 5 Due east). The clear appearance of DNA synapses nether these conditions strongly indicates the presence of nucleoprotein filaments. The electrostatic repulsion between Dna duplexes was probably decreased by bounden of the positively charged basic p53 C‐concluding domain (Fig. iii A) stabilizing shut contacts between Dna duplexes nether the depression salt weather condition (10 mM KCl) ( 41 ).

The above considerations led us to propose the model of p53 C‐terminus bounden to scDNA presented in Figure 6 . Nosotros suggest that each dimer of the tetramer binds one DNA duplex (Fig. 6 A). A similar machinery is proposed for dimeric peptides (Fig. 6 B); ane p53 Deoxyribonucleic acid‐binding region in the dimer binds ane DNA duplex (Fig. 6 B, I) while the 2nd p53 Deoxyribonucleic acid‐bounden region in the dimer interacts with the opposing duplex (Fig. 6 B, II). Since dimeric proteins formed filaments while monomeric proteins did not (Fig. 6 C) nosotros presume that bounden of the kickoff p53 molecule (dimer or tetramer) stabilizes two duplexes in shut proximity, forming an initial complex (Fig. six A and B, II). Further binding of proteins results in formation of higher order structures in which duplex segments are synapsed within long filaments (Fig. 6 A and B, 3). Since multiples of tetramers of wild‐type p53 are plant in solution ( 42 , 43 ) information technology is possible that cooperative Dna–protein and poly peptide–protein interactions contribute to filament germination. In the case of predominantly dimeric forms of p53, filaments could besides arise via simply Dna–protein contacts. It is clear that the binding to opposing duplexes is facilitated by Deoxyribonucleic acid supercoiling to an extent increasing with superhelix density, inasmuch every bit the duplex‐to‐duplex distance varies inversely with superhelix density (reviewed in 37 ).

Since the bacterially expressed full‐length p53b(one–393) also displays filament formation with scDNA of depression superhelix density (D.I.Cherny, G.Brázdová, J.Paleček, E.Paleček and T.1000.Jovin, submitted for publication) and of native superhelix density (D.I.Cherny, unpublished results), we advise that its scDNA binding mode may be similar to that of the C‐terminal segments described hither.

Biological significance of p53–DNA filament formation

Processes such equally DNA recombination and repair, control of supercoiling by topoisomerases and/or factor regulation through DNA looping are accompanied by germination of close contacts between DNA duplexes ( twoscore and references therein). Binding of proteins to such intermediate structures is of critical importance. For example, it was proposed that vaccinia topoisomerase‐mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kb vaccinia genome during virus assembly ( 44 ). We speculate that segments of two aligned chromosomes (exhibiting close contacts between DNA duplexes) could serve as a substrate for p53–Deoxyribonucleic acid filament formation. p53 cadre domain binding to early recombination intermediates like three‐way junctions ( 45 , 46 ) could catalyze formation of an initial nucleoprotein complex. The propagation of filaments (through the C‐terminus) may then block the progression of recombination. Recently, Prabhu et al . ( 47 ) demonstrated that p53 blocks branch migration promoted by proteins such as RuvAB (also as spontaneous branch migration) and modulates the cleavage by Holliday junction resolution proteins such equally RuvC. In addition, inasmuch as p53 C‐terminus tin catalyze strand transfer ( eight , sixteen ) and interacts with Rad51 protein ( 48 ), it is possible that the propagation of p53 filaments could regulate Rad51‐mediated strand transfer. Thus, we propose that the highly efficient germination of nucleoprotein filaments past p53 protein at sites of close contacts betwixt DNA duplexes may play a meaning role in cardinal genetic processes.

ACKNOWLEDGEMENTS

We are indebted to C. Midgley, K. Chiliad. Luciani, T. Halazonetis, S. Gorina, G. Selivanova, M. Protopopova and K. G. Wiman for the supply of plasmids. Technical assistance by Fifty. Karlovska and K. O. Koutska is acknowledged. This work was supported past grants of the Grant Agency of the Czech Republic, no. 301/02/0831 to B.Five. and E.P. and no. 301/99/D078 to J.P., by the Grant Agency of the Academy of Sciences of the Czech Republic, A4004110 to E.P., by the Volkswagen Stiftung, I/72942 to East.P. and T.M.J., past grant NC/5343‐3 of the IGA MH CR to Chiliad.F. The Academy of Sciences of the Czechia supported this work past grant nos Southward 5004009 and Z 5004920.

Figure 1. Purity of p53 deletion mutants isolated from bacterial extracts. ( A ) p53 fragments expressed in bacteria, shown as lines below the map of p53 domains. The evolutionarily conserved domains are indicated: core DNA‐binding domain (shaded; amino acids ∼100–300), tetramerization domain (cross‐hatched; amino acids 325–356) and basic C‐terminal DNA‐binding domain (hatched; amino acids 363–382). Numbers of first and last p53 amino acid residues are indicated for each construct. ( B ) Gel electrophoresis of full‐length p53 and its fragments after their purification by FPLC (see Materials and Methods). Approximately 1 µg of peak fraction of each protein was analyzed by 10–15% gradient SDS–PAGE. Standard protein molecular weight marker (M) was used to compare molecular masses of the proteins.

Effigy 1. Purity of p53 deletion mutants isolated from bacterial extracts. ( A ) p53 fragments expressed in bacteria, shown as lines below the map of p53 domains. The evolutionarily conserved domains are indicated: cadre Dna‐bounden domain (shaded; amino acids ∼100–300), tetramerization domain (cantankerous‐hatched; amino acids 325–356) and bones C‐terminal Dna‐binding domain (hatched; amino acids 363–382). Numbers of start and last p53 amino acid residues are indicated for each construct. ( B ) Gel electrophoresis of full‐length p53 and its fragments afterward their purification by FPLC (see Materials and Methods). Approximately 1 µg of peak fraction of each protein was analyzed by x–15% gradient SDS–PAGE. Standard poly peptide molecular weight marker (Chiliad) was used to compare molecular masses of the proteins.

Figure 1. Purity of p53 deletion mutants isolated from bacterial extracts. ( A ) p53 fragments expressed in bacteria, shown as lines below the map of p53 domains. The evolutionarily conserved domains are indicated: core DNA‐binding domain (shaded; amino acids ∼100–300), tetramerization domain (cross‐hatched; amino acids 325–356) and basic C‐terminal DNA‐binding domain (hatched; amino acids 363–382). Numbers of first and last p53 amino acid residues are indicated for each construct. ( B ) Gel electrophoresis of full‐length p53 and its fragments after their purification by FPLC (see Materials and Methods). Approximately 1 µg of peak fraction of each protein was analyzed by 10–15% gradient SDS–PAGE. Standard protein molecular weight marker (M) was used to compare molecular masses of the proteins.

Effigy 1. Purity of p53 deletion mutants isolated from bacterial extracts. ( A ) p53 fragments expressed in bacteria, shown as lines below the map of p53 domains. The evolutionarily conserved domains are indicated: core DNA‐binding domain (shaded; amino acids ∼100–300), tetramerization domain (cross‐hatched; amino acids 325–356) and basic C‐terminal DNA‐bounden domain (hatched; amino acids 363–382). Numbers of first and concluding p53 amino acid residues are indicated for each construct. ( B ) Gel electrophoresis of full‐length p53 and its fragments after their purification by FPLC (see Materials and Methods). Approximately 1 µg of peak fraction of each poly peptide was analyzed by 10–15% slope SDS–PAGE. Standard poly peptide molecular weight marker (Thou) was used to compare molecular masses of the proteins.

Figure 2. p53 deletion mutants bind scDNA with different selectivities in competition experiments. ( A ) scDNA (0.3 µg; lanes 1 and 2) or linDNA (0.3 µg; lanes 3 and 4) alone was incubated with p53b(1–393) protein (lanes 2 and 4) at a p53 tetramer/DNA molar ratio of 4. A mixture of linear and supercoiled (sc/lin ratio 1:1) forms of pBluescript DNAs containing in total 0.6 µg (lanes 5 and 6), 0.8 µg (lanes 7, 10, 11) or 1.6 µg (lanes 8 and 9) was incubated with p53b(1–393) protein in the molar ratio range 1.5–5.0 protein (tetramer)/DNA (lanes 6 and 8–11). Samples were run on 1.3% agarose gels in 0.33× TBE and stained with ethidium bromide (left). Note that under the conditions used linDNA migrates faster than scDNA. The gels were blotted (right) and p53–scDNA (p53‐sc) and/or p53–linDNA (p53‐lin) complexes were detected using DO‐1 antibody. ( B ) Immunoblots of two representative lanes showing results of the competition assays with the following proteins: lanes 1 and 2, p53(1–393); lanes 3 and 4, p53(1–363); lanes 5 and 6, p53(45–349); lanes 7 and 8, p53(94–312); lanes 9 and 10, p53(44–393); lanes 11 and 12, p53(320–393). Mouse monoclonal antibodies DO‐1 (recognizing amino acids 20–25), DO‐11 (amino acids 176–185), DO‐12 (amino acids 256–270), Bp53‐6.1 (amino acids 381–390), Bp53‐10.1 (amino acids 375–379) and ICA‐9 (amino acids 388–393) were applied to detect epitopes present within the p53 proteins.

Effigy ii. p53 deletion mutants bind scDNA with different selectivities in competition experiments. ( A ) scDNA (0.3 µg; lanes 1 and 2) or linDNA (0.3 µg; lanes 3 and 4) alone was incubated with p53b(one–393) poly peptide (lanes ii and 4) at a p53 tetramer/DNA molar ratio of 4. A mixture of linear and supercoiled (sc/lin ratio 1:1) forms of pBluescript DNAs containing in total 0.vi µg (lanes 5 and half dozen), 0.eight µg (lanes seven, 10, 11) or 1.half-dozen µg (lanes viii and 9) was incubated with p53b(ane–393) poly peptide in the molar ratio range i.5–v.0 poly peptide (tetramer)/DNA (lanes 6 and 8–11). Samples were run on one.three% agarose gels in 0.33× TBE and stained with ethidium bromide (left). Note that nether the conditions used linDNA migrates faster than scDNA. The gels were blotted (right) and p53–scDNA (p53‐sc) and/or p53–linDNA (p53‐lin) complexes were detected using Do‐1 antibody. ( B ) Immunoblots of two representative lanes showing results of the contest assays with the following proteins: lanes 1 and two, p53(ane–393); lanes 3 and 4, p53(1–363); lanes v and 6, p53(45–349); lanes 7 and 8, p53(94–312); lanes nine and 10, p53(44–393); lanes 11 and 12, p53(320–393). Mouse monoclonal antibodies DO‐ane (recognizing amino acids 20–25), Exercise‐eleven (amino acids 176–185), Do‐12 (amino acids 256–270), Bp53‐6.1 (amino acids 381–390), Bp53‐10.i (amino acids 375–379) and ICA‐9 (amino acids 388–393) were practical to notice epitopes present within the p53 proteins.

Figure 2. p53 deletion mutants bind scDNA with different selectivities in competition experiments. ( A ) scDNA (0.3 µg; lanes 1 and 2) or linDNA (0.3 µg; lanes 3 and 4) alone was incubated with p53b(1–393) protein (lanes 2 and 4) at a p53 tetramer/DNA molar ratio of 4. A mixture of linear and supercoiled (sc/lin ratio 1:1) forms of pBluescript DNAs containing in total 0.6 µg (lanes 5 and 6), 0.8 µg (lanes 7, 10, 11) or 1.6 µg (lanes 8 and 9) was incubated with p53b(1–393) protein in the molar ratio range 1.5–5.0 protein (tetramer)/DNA (lanes 6 and 8–11). Samples were run on 1.3% agarose gels in 0.33× TBE and stained with ethidium bromide (left). Note that under the conditions used linDNA migrates faster than scDNA. The gels were blotted (right) and p53–scDNA (p53‐sc) and/or p53–linDNA (p53‐lin) complexes were detected using DO‐1 antibody. ( B ) Immunoblots of two representative lanes showing results of the competition assays with the following proteins: lanes 1 and 2, p53(1–393); lanes 3 and 4, p53(1–363); lanes 5 and 6, p53(45–349); lanes 7 and 8, p53(94–312); lanes 9 and 10, p53(44–393); lanes 11 and 12, p53(320–393). Mouse monoclonal antibodies DO‐1 (recognizing amino acids 20–25), DO‐11 (amino acids 176–185), DO‐12 (amino acids 256–270), Bp53‐6.1 (amino acids 381–390), Bp53‐10.1 (amino acids 375–379) and ICA‐9 (amino acids 388–393) were applied to detect epitopes present within the p53 proteins.

Figure ii. p53 deletion mutants bind scDNA with different selectivities in contest experiments. ( A ) scDNA (0.3 µg; lanes 1 and 2) or linDNA (0.three µg; lanes 3 and four) alone was incubated with p53b(1–393) protein (lanes 2 and 4) at a p53 tetramer/DNA molar ratio of 4. A mixture of linear and supercoiled (sc/lin ratio 1:1) forms of pBluescript DNAs containing in total 0.vi µg (lanes 5 and 6), 0.8 µg (lanes 7, 10, 11) or ane.6 µg (lanes 8 and ix) was incubated with p53b(1–393) poly peptide in the molar ratio range 1.five–5.0 poly peptide (tetramer)/DNA (lanes 6 and 8–11). Samples were run on 1.iii% agarose gels in 0.33× TBE and stained with ethidium bromide (left). Note that under the conditions used linDNA migrates faster than scDNA. The gels were blotted (right) and p53–scDNA (p53‐sc) and/or p53–linDNA (p53‐lin) complexes were detected using Practise‐1 antibody. ( B ) Immunoblots of two representative lanes showing results of the competition assays with the post-obit proteins: lanes 1 and two, p53(ane–393); lanes iii and 4, p53(1–363); lanes 5 and six, p53(45–349); lanes 7 and 8, p53(94–312); lanes 9 and 10, p53(44–393); lanes 11 and 12, p53(320–393). Mouse monoclonal antibodies Practice‐1 (recognizing amino acids 20–25), DO‐eleven (amino acids 176–185), DO‐12 (amino acids 256–270), Bp53‐six.1 (amino acids 381–390), Bp53‐10.1 (amino acids 375–379) and ICA‐ix (amino acids 388–393) were applied to find epitopes nowadays within the p53 proteins.

Figure 3. Amino acid residues 375–378 participate in p53(320–393) binding to scDNA. ( A ) Schematic representation of amino acid residues within the C‐terminal region (amino acids 361–393) of human p53. The basic DNA‐binding domain (amino acids 363–382) and epitopes of each monoclonal antibody used in the supershifting experiments are indicated by lines above and/or below the corresponding sequence. N‐terminal specific antibody DO‐1 (recognizing amino acids 20–25) was used as a negative control. The supershifting experiment ( B ) was performed as follows. First, pBluescript scDNA (0.3 µg) was incubated with p53(320–393) protein (lanes 2–10) at a p53 tetramer/DNA molar ratio = 5. Then, increasing amounts of monoclonal antibodies were added as indicated below the lanes. Samples were run on 1% agarose gels in 0.33× TBE. The gel was blotted ( C ) and developed with rabbit anti‐p53 antibody (see Materials and Methods); only the left four lanes are shown. The presence of p53(320–393) protein in the reaction mixture is indicated by + and/or –, respectively. The positions of p53–scDNA (p53‐sc), immune mAb–p53–scDNA (MAb‐p53‐sc) and mAb–p53 (MAb‐p53) complexes are indicated.

Effigy three. Amino acid residues 375–378 participate in p53(320–393) binding to scDNA. ( A ) Schematic representation of amino acid residues within the C‐last region (amino acids 361–393) of human being p53. The bones DNA‐bounden domain (amino acids 363–382) and epitopes of each monoclonal antibody used in the supershifting experiments are indicated by lines in a higher place and/or below the corresponding sequence. N‐terminal specific antibody DO‐1 (recognizing amino acids 20–25) was used as a negative control. The supershifting experiment ( B ) was performed as follows. Commencement, pBluescript scDNA (0.3 µg) was incubated with p53(320–393) protein (lanes 2–ten) at a p53 tetramer/Deoxyribonucleic acid molar ratio = v. Then, increasing amounts of monoclonal antibodies were added every bit indicated beneath the lanes. Samples were run on i% agarose gels in 0.33× TBE. The gel was blotted ( C ) and developed with rabbit anti‐p53 antibody (see Materials and Methods); only the left 4 lanes are shown. The presence of p53(320–393) protein in the reaction mixture is indicated by + and/or –, respectively. The positions of p53–scDNA (p53‐sc), immune mAb–p53–scDNA (MAb‐p53‐sc) and mAb–p53 (MAb‐p53) complexes are indicated.

Figure 3. Amino acid residues 375–378 participate in p53(320–393) binding to scDNA. ( A ) Schematic representation of amino acid residues within the C‐terminal region (amino acids 361–393) of human p53. The basic DNA‐binding domain (amino acids 363–382) and epitopes of each monoclonal antibody used in the supershifting experiments are indicated by lines above and/or below the corresponding sequence. N‐terminal specific antibody DO‐1 (recognizing amino acids 20–25) was used as a negative control. The supershifting experiment ( B ) was performed as follows. First, pBluescript scDNA (0.3 µg) was incubated with p53(320–393) protein (lanes 2–10) at a p53 tetramer/DNA molar ratio = 5. Then, increasing amounts of monoclonal antibodies were added as indicated below the lanes. Samples were run on 1% agarose gels in 0.33× TBE. The gel was blotted ( C ) and developed with rabbit anti‐p53 antibody (see Materials and Methods); only the left four lanes are shown. The presence of p53(320–393) protein in the reaction mixture is indicated by + and/or –, respectively. The positions of p53–scDNA (p53‐sc), immune mAb–p53–scDNA (MAb‐p53‐sc) and mAb–p53 (MAb‐p53) complexes are indicated.

Figure 3. Amino acid residues 375–378 participate in p53(320–393) binding to scDNA. ( A ) Schematic representation of amino acid residues within the C‐terminal region (amino acids 361–393) of human p53. The basic Dna‐binding domain (amino acids 363–382) and epitopes of each monoclonal antibody used in the supershifting experiments are indicated by lines above and/or below the corresponding sequence. N‐terminal specific antibody DO‐1 (recognizing amino acids 20–25) was used as a negative command. The supershifting experiment ( B ) was performed as follows. First, pBluescript scDNA (0.iii µg) was incubated with p53(320–393) protein (lanes 2–10) at a p53 tetramer/DNA molar ratio = 5. Then, increasing amounts of monoclonal antibodies were added every bit indicated below the lanes. Samples were run on ane% agarose gels in 0.33× TBE. The gel was blotted ( C ) and adult with rabbit anti‐p53 antibody (encounter Materials and Methods); only the left iv lanes are shown. The presence of p53(320–393) protein in the reaction mixture is indicated by + and/or –, respectively. The positions of p53–scDNA (p53‐sc), immune mAb–p53–scDNA (MAb‐p53‐sc) and mAb–p53 (MAb‐p53) complexes are indicated.

Figure 4. p53 oligomeric state of the C‐terminal protein is critical for strong SCS binding. Tetrameric p53(320–393) (lanes 1–3), monomeric p53(361– 393) (lanes 10–12), dimeric mutant p53(319–393)L344A (lanes 4–6) and dimeric fusion GCN4–p53(347–393) (lanes 7–9) were analyzed for selective binding to scDNA in sc/lin (sc/lin ratio 1:1) competition assays (see Materials and Methods). The total amount of DNA was 0.8 µg; protein/DNA molar ratios are indicated. Bound p53 proteins were detected using monoclonal antibodies Bp53‐10.1 and Bp53‐6.1. Three representative lanes for each p53 construct tested are shown.

Figure 4. p53 oligomeric state of the C‐final protein is critical for potent SCS binding. Tetrameric p53(320–393) (lanes one–iii), monomeric p53(361– 393) (lanes 10–12), dimeric mutant p53(319–393)L344A (lanes 4–6) and dimeric fusion GCN4–p53(347–393) (lanes vii–9) were analyzed for selective bounden to scDNA in sc/lin (sc/lin ratio 1:1) contest assays (see Materials and Methods). The full amount of DNA was 0.8 µg; poly peptide/Deoxyribonucleic acid molar ratios are indicated. Bound p53 proteins were detected using monoclonal antibodies Bp53‐10.1 and Bp53‐6.1. 3 representative lanes for each p53 construct tested are shown.

Figure 4. p53 oligomeric state of the C‐terminal protein is critical for strong SCS binding. Tetrameric p53(320–393) (lanes 1–3), monomeric p53(361– 393) (lanes 10–12), dimeric mutant p53(319–393)L344A (lanes 4–6) and dimeric fusion GCN4–p53(347–393) (lanes 7–9) were analyzed for selective binding to scDNA in sc/lin (sc/lin ratio 1:1) competition assays (see Materials and Methods). The total amount of DNA was 0.8 µg; protein/DNA molar ratios are indicated. Bound p53 proteins were detected using monoclonal antibodies Bp53‐10.1 and Bp53‐6.1. Three representative lanes for each p53 construct tested are shown.

Figure four. p53 oligomeric country of the C‐terminal protein is critical for strong SCS binding. Tetrameric p53(320–393) (lanes 1–3), monomeric p53(361– 393) (lanes 10–12), dimeric mutant p53(319–393)L344A (lanes four–6) and dimeric fusion GCN4–p53(347–393) (lanes 7–9) were analyzed for selective binding to scDNA in sc/lin (sc/lin ratio 1:1) contest assays (run across Materials and Methods). The total amount of Deoxyribonucleic acid was 0.8 µg; poly peptide/Deoxyribonucleic acid molar ratios are indicated. Bound p53 proteins were detected using monoclonal antibodies Bp53‐10.1 and Bp53‐6.ane. Iii representative lanes for each p53 construct tested are shown.

Figure 5. Electron microscopy of the protein–DNA complexes formed by different p53 C‐terminal proteins with sc/lin DNA mixture. The panel represents images of the complexes obtained after the incubation of a mixture of supercoiled pBluescript and pBluescript/ Eco RI DNAs (5 nM each) with p53(320–393) ( A ), p53(319–393)L344A ( B ), GCN4–p53(347–393) ( C ) and p53(361–393) ( D ). Protein concentration was 100 nM for a given oligomeric state of the protein in solution (protein/DNA molar ratio = 10). Insets in (A), (B) (upper) and (D) represent magnified views of supercoiled molecules in the presence of proteins. The lower inset in (B) and the inset in (C) represent images of the complexes obtained at a 500 nM concentration of GCN4–p53(347–393) and p53(319–393)L344A proteins, respectively (protein/DNA molar ratio = 50). ( E ) The sc/lin DNA mixture in the absence of proteins; inset, magnified view of supercoiled DNA. Note that under the conditions used for mounting DNA for electron microscopic imaging (10 mM Na HEPES, 10 mM KCl) native supercoiled DNA has a loosely interwound appearance, in accordance with Cherny and Jovin ( 37 ). sc and lin indicate supercoiled and linear DNA, respectively; filaments indicates DNA–protein filaments. For further details see Materials and Methods. The scale bar represents 200 nm.

Effigy v. Electron microscopy of the poly peptide–Deoxyribonucleic acid complexes formed by dissimilar p53 C‐terminal proteins with sc/lin DNA mixture. The console represents images of the complexes obtained subsequently the incubation of a mixture of supercoiled pBluescript and pBluescript/ Eco RI DNAs (5 nM each) with p53(320–393) ( A ), p53(319–393)L344A ( B ), GCN4–p53(347–393) ( C ) and p53(361–393) ( D ). Protein concentration was 100 nM for a given oligomeric state of the poly peptide in solution (poly peptide/Deoxyribonucleic acid tooth ratio = ten). Insets in (A), (B) (upper) and (D) represent magnified views of supercoiled molecules in the presence of proteins. The lower inset in (B) and the inset in (C) represent images of the complexes obtained at a 500 nM concentration of GCN4–p53(347–393) and p53(319–393)L344A proteins, respectively (protein/DNA molar ratio = l). ( E ) The sc/lin DNA mixture in the absence of proteins; inset, magnified view of supercoiled DNA. Note that under the weather used for mounting Dna for electron microscopic imaging (10 mM Na HEPES, 10 mM KCl) native supercoiled Deoxyribonucleic acid has a loosely interwound appearance, in accordance with Cherny and Jovin ( 37 ). sc and lin indicate supercoiled and linear DNA, respectively; filaments indicates DNA–protein filaments. For further details see Materials and Methods. The scale bar represents 200 nm.

Figure 5. Electron microscopy of the protein–DNA complexes formed by different p53 C‐terminal proteins with sc/lin DNA mixture. The panel represents images of the complexes obtained after the incubation of a mixture of supercoiled pBluescript and pBluescript/ Eco RI DNAs (5 nM each) with p53(320–393) ( A ), p53(319–393)L344A ( B ), GCN4–p53(347–393) ( C ) and p53(361–393) ( D ). Protein concentration was 100 nM for a given oligomeric state of the protein in solution (protein/DNA molar ratio = 10). Insets in (A), (B) (upper) and (D) represent magnified views of supercoiled molecules in the presence of proteins. The lower inset in (B) and the inset in (C) represent images of the complexes obtained at a 500 nM concentration of GCN4–p53(347–393) and p53(319–393)L344A proteins, respectively (protein/DNA molar ratio = 50). ( E ) The sc/lin DNA mixture in the absence of proteins; inset, magnified view of supercoiled DNA. Note that under the conditions used for mounting DNA for electron microscopic imaging (10 mM Na HEPES, 10 mM KCl) native supercoiled DNA has a loosely interwound appearance, in accordance with Cherny and Jovin ( 37 ). sc and lin indicate supercoiled and linear DNA, respectively; filaments indicates DNA–protein filaments. For further details see Materials and Methods. The scale bar represents 200 nm.

Figure 5. Electron microscopy of the poly peptide–DNA complexes formed by dissimilar p53 C‐terminal proteins with sc/lin Dna mixture. The panel represents images of the complexes obtained subsequently the incubation of a mixture of supercoiled pBluescript and pBluescript/ Eco RI DNAs (five nM each) with p53(320–393) ( A ), p53(319–393)L344A ( B ), GCN4–p53(347–393) ( C ) and p53(361–393) ( D ). Protein concentration was 100 nM for a given oligomeric state of the protein in solution (protein/Dna molar ratio = 10). Insets in (A), (B) (upper) and (D) represent magnified views of supercoiled molecules in the presence of proteins. The lower inset in (B) and the inset in (C) correspond images of the complexes obtained at a 500 nM concentration of GCN4–p53(347–393) and p53(319–393)L344A proteins, respectively (poly peptide/DNA molar ratio = l). ( E ) The sc/lin DNA mixture in the absence of proteins; inset, magnified view of supercoiled DNA. Note that under the atmospheric condition used for mounting DNA for electron microscopic imaging (10 mM Na HEPES, ten mM KCl) native supercoiled DNA has a loosely interwound appearance, in accordance with Cherny and Jovin ( 37 ). sc and lin signal supercoiled and linear Deoxyribonucleic acid, respectively; filaments indicates Dna–poly peptide filaments. For further details see Materials and Methods. The scale bar represents 200 nm.

Figure 6. A model of p53 C‐terminus interaction with two DNA helices of scDNA. The C‐terminal fragment of p53, consisting of a DNA‐binding domain (black circles) and an oligomerization domain (empty formations, shown for predominant tetrameric or dimeric protein forms, respectively) binds single DNA duplexes (stage I). Unbound DNA‐binding domains of p53 tetramers or dimers are capable of interacting further with an opposing DNA duplex, located close to the first one, due to the plectonemic configuration of scDNA [stage II in ( A ) and ( B )], thus stabilizing DNA strands in close proximity. Then, the next p53 molecule binds opposing DNA duplexes, leading to the synapsing of two non‐contiguous duplexes, imaged as a long DNA–protein filament [stage III in ( A ) and ( B )]. Bound proteins can be positioned either in juxtaposed registers due to protein–protein interactions (cooperative binding, the most probable mode for protein tetramers) or separately (the most probable mode for protein dimers). Both modes of positioning of bound proteins may occur concurrently within a single filament. Monomeric p53 binds DNA duplex but does not manifest protein–protein interactions either in solution or in the bound state. Hence, no filaments can be produced ( C ).

Effigy vi. A model of p53 C‐terminus interaction with two DNA helices of scDNA. The C‐terminal fragment of p53, consisting of a Dna‐bounden domain (black circles) and an oligomerization domain (empty formations, shown for predominant tetrameric or dimeric protein forms, respectively) binds single DNA duplexes (stage I). Unbound Dna‐binding domains of p53 tetramers or dimers are capable of interacting further with an opposing DNA duplex, located shut to the commencement one, due to the plectonemic configuration of scDNA [stage Two in ( A ) and ( B )], thus stabilizing Dna strands in shut proximity. Then, the next p53 molecule binds opposing Deoxyribonucleic acid duplexes, leading to the synapsing of two not‐contiguous duplexes, imaged as a long Dna–protein filament [phase Three in ( A ) and ( B )]. Bound proteins can be positioned either in juxtaposed registers due to protein–protein interactions (cooperative binding, the nigh probable fashion for protein tetramers) or separately (the most probable mode for protein dimers). Both modes of positioning of bound proteins may occur concurrently within a unmarried filament. Monomeric p53 binds DNA duplex merely does not manifest protein–protein interactions either in solution or in the leap state. Hence, no filaments tin exist produced ( C ).

Figure 6. A model of p53 C‐terminus interaction with two DNA helices of scDNA. The C‐terminal fragment of p53, consisting of a DNA‐binding domain (black circles) and an oligomerization domain (empty formations, shown for predominant tetrameric or dimeric protein forms, respectively) binds single DNA duplexes (stage I). Unbound DNA‐binding domains of p53 tetramers or dimers are capable of interacting further with an opposing DNA duplex, located close to the first one, due to the plectonemic configuration of scDNA [stage II in ( A ) and ( B )], thus stabilizing DNA strands in close proximity. Then, the next p53 molecule binds opposing DNA duplexes, leading to the synapsing of two non‐contiguous duplexes, imaged as a long DNA–protein filament [stage III in ( A ) and ( B )]. Bound proteins can be positioned either in juxtaposed registers due to protein–protein interactions (cooperative binding, the most probable mode for protein tetramers) or separately (the most probable mode for protein dimers). Both modes of positioning of bound proteins may occur concurrently within a single filament. Monomeric p53 binds DNA duplex but does not manifest protein–protein interactions either in solution or in the bound state. Hence, no filaments can be produced ( C ).

Figure vi. A model of p53 C‐terminus interaction with two DNA helices of scDNA. The C‐terminal fragment of p53, consisting of a Deoxyribonucleic acid‐binding domain (black circles) and an oligomerization domain (empty formations, shown for predominant tetrameric or dimeric protein forms, respectively) binds unmarried Deoxyribonucleic acid duplexes (stage I). Unbound Deoxyribonucleic acid‐binding domains of p53 tetramers or dimers are capable of interacting further with an opposing DNA duplex, located close to the start one, due to the plectonemic configuration of scDNA [stage Two in ( A ) and ( B )], thus stabilizing Dna strands in close proximity. Then, the side by side p53 molecule binds opposing DNA duplexes, leading to the synapsing of two non‐contiguous duplexes, imaged as a long Dna–protein filament [stage III in ( A ) and ( B )]. Leap proteins can be positioned either in juxtaposed registers due to protein–poly peptide interactions (cooperative binding, the nigh probable way for poly peptide tetramers) or separately (the most probable mode for protein dimers). Both modes of positioning of leap proteins may occur concurrently within a unmarried filament. Monomeric p53 binds Dna duplex but does non manifest poly peptide–protein interactions either in solution or in the bound state. Hence, no filaments tin can be produced ( C ).

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