Proc. Natl. Acad. Sci. USAVol. 90, pp. 3319-3323, April 1993Biochemistry

The p53 protein is an unusually shaped tetramer that binds directlyto DNA

(tumor suppressor/chemical crosslinking/gel riltration/sucrose gradients/DNA-binding protein)

PAULA N. FRIEDMAN*, XINBIN CHEN, JILL BARGONETTI, AND CAROL PRIVEStDepartment of Biological Sciences, Columbia University, New York, NY 10027

Communicated by Bert Vogelstein, December 29, 1992

ABSTRACT We have analyzed the size and structure ofnative immunopurified human p53 protein. By using a com-bination of chemical crosslinking, gel ifitration chromatogra-phy, and zonal velocity gradient centrifugation, we have de-termined that the predominant form of p53 in such prepara-tions is a tetramer. The behavior of purified p53 in gels andsucrose gradients implies that the protein has an extendedshape. Wild-type p53 has been shown to bind specifically tosites in cellular and viral DNA. We show in this study bySouthwestern ligand blotting and by analysis of DNA-boundcrosslinked p53 that p53 monomers, dimers, and tetramers canbind directly to DNA.

As alterations in the function of the p53 gene product may beinvolved in a wide variety of human cancers, it is imperativeto gain insight into this important protein. p53 contains fourhighly conserved regions that are 90-100% homologousamong the diverse vertebrate species examined (1). Strik-ingly, the great majority of missense mutations identified indiverse human tumor types are located in the conservedblocks within p53 (reviewed in ref. 2). These data suggest thatthese highly conserved regions of p53 are crucial for thecorrect functioning of the wild-type protein. In addition to theimportance of the conserved regions of p53, key features ofthe p53 polypeptide reside in its more peripheral regions aswell. The NH2 portion of the p53 protein was shown toinclude a strong transcriptional activation domain (3-6). TheCOOH portion contains both primary and secondary nuclearlocalization signals (7, 8), as well as sequences that contributeto p53 protein-protein interactions (9, 10) and protein-DNAinteractions (11).

Important clues to the function of p53 are derived fromstudies showing that p53 binds site-specifically to DNA(12-16). Furthermore, p53 was shown to directly activatetranscription in vivo (15, 17-19) and in vitro (20) in a mannerthat depends upon sequence-specific interactions with thetranscription template. Defining the native size and shape ofthe p53 molecule and the forms of p53 that bind to DNA orto other proteins is likely to provide new insights into itsfunction.

MATERIALS AND METHODSPurification of p53 Proteins. Recombinant baculoviruses

expressing human wild-type and mutant (His-273) p53 havebeen described (21). Extracts of infected Sf21 insect cellswere prepared and p53 was purified from lysates by immu-noaffinity procedures (22).

Crosslinking Analysis. Proteins were incubated in the pres-ence or absence of 0.01% or 0.1% glutaraldehyde for 15 min

at 37°C before analysis by SDS/PAGE or sucrose gradientcentrifugation.

Gel Filtration Chromatography. p53 protein (25 ,g) wasapplied to a TSK 3000 gel filtration column previouslyequilibrated with column buffer (100 mM sodium phosphate,pH 7.07/1 mM EDTA) and run on the Pharmacia FPLCsystem. Elution was carried out at 30 ml/hr, and elutedsamples were collected in 1-ml fractions and absorbancereadings were confirmed by both SDS/PAGE and Bio-Raddye-binding protein assay. Protein molecular weight stan-dards (1 mg/ml) were run and analyzed in parallel.

Sucrose Gradient Centrifugation. Immunoaffinity-purifiedp53 (2 ,g) in 500 ,ul of 50 mM Tris-HCl, pH 8.0/150 mMNaCl/1% (wt/vol) Nonidet P-40/1 mM dithiothreitol waslayered directly on a gradient of 5-20% (wt/vol) sucrose (5ml) in phosphate-buffered saline (PBS: 8 mM Na2HPO4/1.5mM KH2PO4/140 mM NaCl/3 mM KCl, pH 7.5). Gradientswere centrifuged in a Beckman SW50.1 rotor at 25,000 rpmfor 16 hr at 4°C. Fractions (-300 IlI) were collected andsubjected to SDS/PAGE after precipitation with 5% (wt/vol)trichloroacetic acid in the presence of 20 ,ug of bovine serumalbumin as carrier. Proteins were electrotransferred to nitro-cellulose and immunostained with a combination of p53-specific antibodies Pab 421 and Pab 1801.DNA Binding. Southwestern analysis of p53 proteins was

as described (11). Duplicate samples of proteins were sub-jected to SDS/10% PAGE and electrotransferred to nitro-cellulose filters. Half ofeach filter was probed with Pab 1801.The other half was washed overnight in standard bindingbuffer [SBB: 10 mM Tris HCl, pH 7.0/50 mM NaCl/l mMEDTA/0.02% bovine serum albumin (BSA)/0.02% polyvi-nylpyrrolidone/0.02% Ficoll]. It was then incubated with 100ng of 32P-end-labeled double-stranded oligodeoxynucleotidecorresponding to the primary binding region for p53 on simianvirus 40 (SV40) DNA (nt 34-73) in a heat-sealed bag for 3 hrat 20°C with nonspecific DNA (pBR322, 10 ug/ml) in SBB.The blot was washed three times for 20 min with SBB andthen dried and exposed to x-ray film.For DNA binding in solution, purified p53 proteins were

incubated with the 32P-labeled SV40 oligonucleotide for 15min, as described (13), and then incubated with glutaralde-hyde for an additional 15 min. Samples were subjected toSDS/5-10% PAGE and subsequent Western immunoblottingwith Pab 1801, after which the blots were dried and exposedto x-ray film.

RESULTSCrosslinking Experiments Show That p53 Preferentially

Forms Tetramers. Stenger et al. (23) used protein crosslinkinganalysis to show that murine p53 assembles preferentially

Abbreviations: BSA, bovine serum albumin; SV40, simian virus 40.*Present address: Department of Clinical Immunology and Biolog-ical Therapy, M.D. Anderson Cancer Center, Houston, TX 77030.tTo whom reprint requests should be addressed.

3319

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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3320 Biochemistry: Friedman et al.

into multimeric forms that are most likely tetramers. Usingsimilar conditions, we incubated immunopurified human p53with 0.01% or 0.1% glutaraldehyde and then subjected themixtures to SDS/PAGE and silver staining (Fig. 1 Upper,lanes A-C). When incubated with 0.01% glutaraldehyde, p53displayed forms that migrated as the monomer and at leasttwo species migrating in the vicinity of (but more slowly than)the 92-kDa marker (lane B). The mobilities of these formsrelative to the marker proteins suggested that they were p53dimers. The p53 dimer form appeared consistently as adoublet: this suggests either that p53 consists of two distinctsubpopulations or, alternatively, that p53 has more than onedeterminant of dimerization. Additionally, small amounts offorms migrating at :200 kDa were detected under theseconditions. It is not known whether or not the doubletmigrating slightly more rapidly than the 200-kDa markerconsisted of p53 trimers. At the higher concentration ofglutaraldehyde (0.1%), smaller forms of p53 were convertedto a predominant species migrating close to the 200-kDamarker polypeptide (lane C). This suggests strongly that thisform was a tetramer. After treatment with 0.1% glutaralde-hyde, some p53 failed to enter the gel, suggesting that stilllarger forms had been crosslinked. Similar results wereobtained with another crosslinking reagent, dimethyl pime-limidate (data not shown). We also compared crosslinked p53

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with a comparable amount of similarly treated BSA, whichnormally migrates in SDS/polyacrylamide gels as ~'66 kDa(lanes D-F). In contrast to what we observed with p53 at0.01% glutaraldehyde, only a small proportion of monomerBSA was converted to the dimer (lane E), while at 0.1%glutaraldehyde a rather diffuse ladder of BSA forms wasgenerated most likely representing monomer, dimer, trimer,etc., rather than a single unique species (lane F). Thus,glutaraldehyde treatment produced strikingly different p53and BSA crosslinked oligomers. The more extensivelycrosslinked forms of both proteins migrated more rapidlythrough the gel than would be expected on the basis ofmolecular mass, most likely because of their more compactshape. Although, somewhat unexpectedly, crosslinked oligo-mers were more effectively silver stained, no such differ-ences in the immunoreactivity of p53 monomers or largerforms were detected (e.g., see Fig. 5). The relative mobilitiesofthe crosslinked forms ofthese two proteins were comparedin three gels each containing different proportions of poly-acrylamide (Fig. 1 Lower). In each case the migration of themajor p53 crosslinked form relative to the BSA forms wassimilar, supporting the likelihood that it was a tetramer. Ourdata with crosslinked human p53 thus confirm prior resultsobtained with murine p53 (23) and indicate that a tetramericform of p53 is preferentially stabilized.

Gel Filtration and Zonal Velocity Centrifugation AnalysesSuggest That p53 Forms Tetramers. To analyze further thenative size of p53, the protein was subjected to FPLC gelfiltration on a TSK 3000 column (Fig. 2). The elution profilewas compared with that ofprotein standards. The majority ofthe p53 protein appeared at an elution volume close to that ofthe 440-kDa apoferritin standard. A minor "shoulder" to thismajor form and small amounts of a species that was eluted atvolume positions between those of the 44- and 154-kDamarkers were also apparent. There were also detectablequantities of p53, representing <20% of the total protein, inthe void volume of the column, suggesting the existence ofstill larger forms of p53 (data not shown). We estimated thatthe Stokes radius of the major form of immunopurified p53was 64 A, by comparison with the elution volumes of severalstandard proteins of known Stokes radii that were run inparallel with p53.Because the major form of p53 from gel exclusion columns

comigrated with the apoferritin standard and thus appeared to

0.06

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0.02-FIG. 1. p53 is crosslinked preferentially into tetramers. (Upper)

Purified wild-type human p53 (1 ,ug) (lanes A-C) or BSA (1 ,g) (lanesD-F) were incubated with 0o (lanes A and D), 0.01% (lanes B andE), or 0.1% (lanes C and F) glutaraldehyde for 15 min at 37°C.Samples were analyzed by SDS/5-10% PAGE followed by silverstaining. Molecular masses of marker proteins (lanes M) in kilodal-tons are indicated. (Lower) Purified p53 and BSA (1 ,ug in each case)were treated with 0.1% glutaraldehyde for 15 min and then analyzedby SDS/PAGE in gels containing gradients of5-20% (E), 5-12% (O),or 5-10o (v) polyacrylamide. The migration of the crosslinked p53in each case (indicated by arrows) was compared with that of thecrosslinked BSA monomers (66 kDa), dimers (132 kDa), trimers (198kDa), and tetramers (264 kDa).

0.01

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FIG. 2. Gel filtration profile of p53. Purified wild-type p53 (5 Ag)was applied to a TSK 3000 column and eluted with column buffer intwenty-five 1-ml fractions. Arrows denote the elution positions ofmarker proteins run in a parallel experiment: thyroglobulin, 669 kDa(Stokes radius, 88 A); ferritin, 440 kDa (61 A); catalase, 232 kDa(52.2 A); aldolase, 154 kDa (48.1 A); ovalbumin, 43 kDa (30.5 A).

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Proc. Natl. Acad. Sci. USA 90 (1993)

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be considerably larger than a tetramer, we used sucrosegradient sedimentation analysis as an additional method ofsize determination (Fig. 3A). Unexpectedly, the majority ofthe p53 sedimented rather slowly, at a position close to thetop of the gradient, between the positions of 44-kDa (3.7S)and 158-kDa (7.8S) protein markers run in parallel gradients,suggesting that the major p53 species was a monomer ordimer (or both). This experiment when repeated several timesunder different conditions provided essentially similar results(data not shown). By comparing the sedimentation of p53with proteins of established s values run in parallel sucrosegradients, we concluded that the major p53 species had asedimentation coefficient of -6.5 S.One explanation for the disparity in apparent molecular

mass ofp53 as determined by gel electrophoresis and gradientcentrifugation is that the larger oligomeric forms of p53 areunstable and dissociate when centrifuged through a sucrosegradient, a phenomenon for which there is established prec-edent (24). However, evidence that the major slowly sedi-menting form of p53 detected after density gradient sedimen-tation does not reflect the true size of p53 and is, in fact,oligomeric was derived from an experiment in which p53 wassubjected to sucrose gradient centrifugation either without orwith prior crosslinking by glutaraldehyde (Fig. 3 B and C).We used the higher amount of glutaraldehyde (0.1%) asshown in Fig. 1 and confirmed by SDS/PAGE that nomonomer and dimer p53 forms were present after this treat-ment and that only the -200-kDa p53 species was detectable(data not shown). Such treated or untreated p53 preparationswere analyzed by sucrose gradient centrifugation followed bySDS/PAGE and Western blotting with the p53-specificmonoclonal antibody Pab 1801. The p53 in each fraction wasthen quantitated by densitometry. The untreated and thecrosslinked p53 proteins displayed virtually identical sedi-mentation through the sucrose gradient. This result effec-

tively ruled out the possibility that p53 multimers are unstableand dissociate during sucrose gradient centrifugation.

Siegel and Monty (25) demonstrated that the molecularweight and fractional coefficient of several proteins could beestablished from their Stokes radii derived from gel filtrationand sedimentation coefficients obtained from sucrose gradi-ent centrifugation. We applied the equation

[1]6(r1-Nas

M=(1--Vp)

whereMis the molecular mass, a is the Stokes radius, s is thesedimentation coefficient, v is the partial specific volume(value used, 0.725), q is the viscosity of the medium (valueused, 1), p is the density of the medium (value used, 1), andN is Avogadro's number.

This led to an estimate of the molecular mass of the majorform ofp53 as 171 kDa. Although in SDS/PAGE p53 migratesto a position approximately intermediate between those ofthe 43- and 68-kDa marker polypeptides, the mass of its 393constituent amino acids is 44 kDa, and therefore the mass ofa p53 tetramer would be 176 kDa which is close to the valuederived from Eq. 1. Accordingly, by combining the results ofthe crosslinking experiment and the molecular mass of themajor form of p53 derived from gel filtration and gradientsedimentation, we conclude that the unit multimer of p53 isa tetramer. The seemingly paradoxical behavior ofp53 in gelsand sucrose gradients also suggests an extended shape of thetetramer. By calculating the frictional coefficient of p53 (25)as

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FIG. 3. Sucrose gradient anal-ysis of p53. Purified wild-type hu-man p53 (2 ,ug) was centrifugedthrough 5-20%o sucrose gradients.Western blotting of gradient frac-tions is shown in A. Lane 1 rep-resents the top and lane 15 thebottom of the gradient. The posi-tions in molecular masses (kDa) ofnative protein standards centri-fuged through parallel sucrose gra-dients are indicated at top. Theelectrophoretic migration of stan-dards is shown at left. Purifiedhuman p53 (1 ,ug) was also incu-bated in the absence (B) or pres-ence (C) of 0.1% glutaraldehydefor 15 min at 37°C prior to centrif-ugation through a 5-20% sucrosegradient and processing of frac-tions as described in A. The rela-tive positions of molecular massstandards in B and C were identi-cal to those shown in A. p53 in thefractions shown in B and C wasquantified with a Biolmage Visage110 densitometer.

A

200 :>

92 s

68

44V

4 3 2 1

Biochemistry: Friedman et al.

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3322 Biochemistry: Friedman et al.

we have obtained an approximate f/fo value of 1.75. Thisvalue suggests that p53 is more ellipsoid than spherical.Wild-Type p53 Binds Directly to DNA. Although by several

criteria immunopurified p53 is a DNA-binding protein, apoint ofconcern is the possibility that p53 itself does not bindDNA, but that a minor but potent unrelated component in theimmunopurified wild-type p53 preparations is actually theDNA-binding entity. To address these issues, p53 binding toDNA was examined by Southwestern analysis in whichequivalent amounts of either wild-type or His-273 mutanthuman p53 proteins were resolved by SDS/PAGE and elec-trotransferred to nitrocellulose. The resulting blot was thendivided so that half contained p53 samples that were identi-fied by immunostaining with the p53-specific antibody Pab421 (Fig. 4A). The remaining half was incubated with a32P-labeled duplex fragment that contains the p53 binding siteon SV40 (13, 16), washed, and exposed to x-ray film (Fig. 4B).The results of this experiment were unequivocal: only the p53polypeptide in our preparations of immunopurified proteinbound to the DNA. Furthermore, the wild-type p53 boundDNA with far greater efficiency than the mutant p53. Whenthe ability of a normal and a mutated version of a strongcellular p53 binding sequence (RGC, refs. 12 and 16) werecompared for their ability to bind to p53 by this analysis, wefound that the immobilized renatured p53 bound -3-foldmore of the normal version than of the mutant version of thisoligonucleotide (data not shown). Thus, using the Southwest-ern binding protocol we have been unable to identify condi-tions for demonstrating a marked difference between the twosequences, suggesting the possibility that the protocol mea-sures nonspecific binding more effectively than it does spe-cific binding.

Crosslinked p53 Forms Are Bound to DNA. To furtheranalyze the interactions of p53 with DNA, we combined aDNA-binding procedure with the protein-crosslinking proto-col shown in Fig. 1. p53 was incubated first without (Fig. SA)or with (Fig. 5 B and C) the 32P-labeled duplex SV40oligonucleotide and then with 0.01% or 0.1% glutaraldehyde.The samples were heated in SDS sample buffer and subjectedto SDS/PAGE. Nitrocellulose blots were then probed for p53(Fig. 5 A and B) and exposed to x-ray film (Fig. SC). At thehigher concentration of glutaraldehyde, when incubated inthe presence of the DNA, a new, still larger form of p53 wasdetected (indicated by arrowhead). When the blot was ex-posed to film the resulting autoradiogram indicated that DNAwas associated with all forms of p53. From comparison oftheamount of immunoreactive p53 detected on the Western blotin Fig. SB with the autoradiogram in Fig. SC it is evident thatthe monomer and dimer, as well as the tetramer and the new

M 1 2 M 1 2

92-68-

45-

30b

A B

FIG. 4. p53 binds directly to DNA. Duplicate samples of His-273mutant (lanes 1) and wild-type (lanes 2) p53 proteins were subjectedto SDS/Ilo PAGE and electrotransferred to nitrocellulose. Half ofthe Western blot was probed with monoclonal antibody Pab 1801 (A).The other half was incubated with the SV40 32P-labeled duplexoligonucleotide in standard binding buffer. This blot was washed,dried, and exposed to x-ray film (B). Lanes M, standard proteinmarkers.

200

92 -

68

45 x

30

M a bc d e f d e f

_ ]..6 ]4

1 2

A B C

FIG. 5. Crosslinked p53 monomers, dimers, and tetramers bindDNA. Immunopurified wild-type human p53 was incubated in theabsence (A) or presence (B and C) of 20 ng (lanes d) or 40 ng (lanese and f) of 32P-labeled oligonucleotide as described in Fig. 4.Reactions were then treated with 0o (lane a), 0.01% (lanes b, d, ande), or 0.1% (lanes c and f) glutaraldehyde for 15 min as in Fig. 1.Samples were analyzed by SDS/10o PAGE and then subjected tosubsequent Western blotting with monoclonal antibody Pab 1801 (Aand B). The blot shown in B was exposed to x-ray film (C). Positionsof standard polypeptide markers with molecular masses in kilodal-tons are shown at left. Positions of DNA-bound and non-bound p53monomers, dimers, and tetramers, indicated by 1, 2, and 4 respec-tively, are bracketed at right.

larger form, can bind to the oligodeoxynucleotide. Indeed, asp53 protein could not be detected by immunostaining at thepositions of either the p53 monomer or of the p53 dimerbound to the oligonucleotide (compare lanes d and e in Fig.5B with lanes d and e in Fig. SC), these data suggest that p53monomer and dimer bind efficiently to DNA. Clearly, boththe tetramer and the larger p53 form are also capable ofbinding well to DNA. We conclude that the major form ofimmunopurified p53 is a tetramer and that monomers throughtetramers and even multiples of tetramers are the DNA-binding forms of p53.

DISCUSSIONWe have used several methods to determine the structure ofpurified, native p53 protein. It is possible to combine theresults obtained into a fairly cohesive framework and toconclude that human p53 consists of a series of discreteoligomers of which the most abundant is apparently a tetra-mer with extended shape. Our data agree with previousstudies indicating that p53 in mammalian cells is oligomeric(26, 27) and that p53 polypeptides from different species oreven different genetics from within the same species associ-ate with one another (9, 10, 21, 28). Confirming observationsfrom Tegtmeyer's group (23), we found that glutaraldehydeinduced the efficient formation of a species ofhuman p53 thatmigrated in the vicinity of the 200-kDa weight marker inSDS/PAGE. We have also analyzed both native andcrosslinked p53 in nondenaturing polyacrylamide gels andhave observed, as did Stenger et al. (23), that the proteinmigrates as a series of large forms that may representtetramers and multiples of tetramers. The most rapidly mi-grating form of human p53 appears in the vicinity of a=270-kDa marker polypeptide (data not shown).Gel filtration resulted in the detection of a major form of

p53 that was eluted at a similar position as a 440-kDa proteinstandard, suggesting a multimer larger than a tetramer. Twominor forms of p53 that comigrated with protein standards ofsmaller molecular mass were also resolved by gel filtration.Evidence supporting our assumption that the major form is atetramer was derived from an experiment showing thatcrosslinked p53 tetramers were coeluted with the 440-kDastandard protein (data not shown). Furthermore, crosslinked

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p53 dimers were eluted at a position equivalent to theshoulder of the main peak shown in Fig. 2.The majority of p53 sedimented slowly through sucrose

gradients, as if it were a monomer or a dimer. How can weexplain the apparent discrepancy between the results ob-tained with nondenaturing gels or gel exclusion chromatog-raphy and those with sucrose gradients? If p53 oligomerswere elongated rather than spherical or globular, we mightpredict that their migration would be retarded in all the abovesystems. There are precedents for proteins that displayanomalous behavior when different approaches to size de-termination are used. For example the Escherichia coli DNApolymerase III holoenzyme subunit Tau protein (29) and thereovirus 1 cell attachment protein (30) display somewhatcontradictory behavior when analyzed by gel filtration anddensity gradient. The value of 1.75 obtained for the frictionalcoefficient of p53 strongly supports the suggestion that thep53 tetramer has an elongated rather than spherical shape.However, more definitive characterization of the unit form ofp53 awaits further analysis such as electron microscopy and,ultimately, x-ray crystallography.The sequence-specific DNA-binding properties of p53 may

provide a potentially important clue to its function in cells,especially when coupled with the observation that, whenbound to DNA, p53 activates transcription. Therefore un-derstanding the structure of p53 as it binds to DNA is ofimportance. Our DNA-binding experiments show that p53monomers through tetramers and still larger forms binddirectly to DNA. While it is not possible to conclude fromthese experiments whether or not the DNA binding detectedis sequence-specific, additional methods have been used todemonstrate that p53 binds specifically to DNA (12, 13, 16).Several p53 binding sites have been identified, and a recentcompendium of several such sequences has revealed thatmany p53 binding regions contain two symmetrical 5'-RRRC(A/T)(T/A)GYYY-3' sites spaced 0-13 bp apart (14).These data fit well with our results that p53 is a highlyelongated tetramer and that all forms of p53 bind directly toDNA. It can be speculated that a p53 dimer binds onesymmetrical site and that p53 dimers may be a stable inter-mediate form of the protein. However, tetramers are the mainspecies detected in the absence of DNA and also appear toform preferentially under protein crosslinking conditions. Wehave analyzed several p53 sites by DNase I protectionanalysis, which revealed strongly protected regions borderedby alternating patterns of DNA protection and hypercuttingsuggesting wrapping of DNA by p53 (13, 15, 16). Thesecharacteristics are consistent with the likelihood that p53possesses an extended and flexible shape.

Confirming earlier reports (12-15, 17), in this study weshow that the His-273 mutant p53 binds DNA very poorly.Since we have failed to note any significant differences in thebehavior of the wild-type or His-273 mutant p53 aftercrosslinking analysis, sucrose gradient centrifugation, ornondenaturing gel analysis (data not shown), this suggeststhat oligomerization of the mutant protein is not related to itsDNA-binding defect. Virtually all tumor-derived mutant p53proteins were shown to exhibit striking defects in DNAbinding (31), suggesting that altering this activity of p53,rather than oligomerization, is related to the process ofoncogenesis. Indeed, since, as shown here, p53 monomerscan bind to DNA, the identification of means to disrupt p53tetramers might prove fruitful in overcoming the dominantnegative effect of many mutant forms of p53.

We are grateful to P. Tegtmeyer and to V. Rotter for communi-cating the results of p53 crosslinking and DNA-binding experimentsprior to publication. We thank B. Vogelstein for stimulating discus-sions about the DNA-binding properties of p53 which provided theimpetus for some of these experiments. Additionally, C. Siegall andA. Wahl gave assistance and advice regarding sizing analysis. EllaFreulich provided outstanding technical assistance. This work wassupported by National Institutes of Health Grant CA33620.

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