Binding of p53DBD to p53 sites as a function of the CWWG motif
The CWWG region is the most conserved region of p53 REs (44
), even though the WW dinucleotide is uncontacted in complex with p53DBD (12
). To probe the role of the non-contacted central dinucleotide in p53/DNA interactions, we studied the effect of changing the central step on p53DBD binding affinity to these targets. We started with the sequence GGGCATGTCC (Con1, ), because it is based on a decamer sequence that was identified by in vitro
selection experiments as a strong p53-binding site (5
), with high transactivation activity in vivo
). We then changed the middle dinucleotide within each half-site to T–A (Con2, ) or A–A (Con3, ). The results of EMSAs with these sequences are shown in . Surprisingly, a major variability in this series was the stoichiometry of the binding interaction, i.e. the oligomeric state of p53DBD in the complexes formed with each target. Con1 showed two distinct shifted bands, a dominant upper band and a minor lower band, similar to those displayed by the sequences studied by Kitayner et al
), whereas Con2 and Con3 show only the major upper band. Gel-mobility-shift assays are unique in their ability to distinguish between complexes that differ in their oligomeric composition (37
Figure 1. Binding affinity measurements by EMSA of p53 consensus REs as a function of the central CWWG motif. (A) Con1, with a central CATG motif. (B) Con2, with CTAG. (C) Con3, with CAAG. DNA targets were imbedded in hairpin constructs (concentration <0.1nM). (more ...)
To quantify these gels with confidence, and for each species separately, we must first determine the exact binding stoichiometry of each complex in the gel. The stoichiometry was determined using the continuous variation (Job plot) assay (33
). In this method, the total molar concentration of protein and DNA is held constant, whereas their molar ratios are continuously varied, from only DNA to almost only protein (A). The ratio at which the bound complex is maximal is the binding stoichiometry for that complex. The reaction was carried out using the Con1 sequence with the total molar concentration fixed at 600
nM, which is thus significantly above the dissociation constant of the complex (). Approximating the experimental points by two lines of limiting slopes (B) produced a stoichiometry of 1:3.94 (0.06) for the upper bound complex and 1:1.94 (0.08) for the lower bound complex, which established that the upper bound complex is a tetramer of p53DBD and the lower one is a dimer of p53DBD bound to DNA. A proof for cooperative binding is when the maximum in the Job plot (and hence the resultant stoichiometry) is independent of the total protein and DNA concentration (33
). Hence, we repeated the experiment at 900
nM and at 1200
nM total molar concentration (Supplementary Figure S1
). The resulting stoichiometry was 1:4.0 (0.2) and 1:1.96 (0.07) at 900
nM, and 1:4.1 (0.2) and 1:2.1 (0.2) at 1200
nM, for the upper and the lower bound bands, respectively, thus establishing that p53DBD binds cooperatively to its REs, whether the binding is as a tetramer (upper bound band) or as a dimeric species (lower bound band).
Figure 2. Continuous variation analysis of p53DBD binding to Con1. (A) A representative EMSA gel of p53DBD binding to Con1 (of three independent experiments). Total macromolecular concentration was fixed at 600nM. The mole fraction of protein, shown below (more ...)
Another convenient method to easily check the binding stoichiometry is to mix together two proteins constructs differing only in their molecular weight (47
). Therefore, to further elucidate the nature of the different binding species we ran an EMSA gel with the Con1 target, using an equimolar mixture of two different p53 constructs: p53DBD (amino acids 94–293) and p53TD (amino acids 1–290). Supplementary Figure S2A
shows that p53TD formed a complex with Con1 that migrates higher on the gel than the complex containing p53DBD, and that only a single complex band is observed. Supplementary Figure S2B
shows that upon mixing the two proteins, the upper band is split into three separate bands, of which the lower one is the prominent one. This confirmed that the uppermost shifted band with Con1 and p53DBD is a tetramer, and that this tetramer is composed of tightly held dimers. Mixing of independent monomeric species would have resulted in a split to five individual bands. It also confirmed that the lower shifted band with Con1 and p53DBD is not a tetramer. The N-terminal domain is known to have an inhibitory effect on the binding of the DBD to p53 targets (48
), which may be the reason why p53TD is not stably bound as a dimer. This is also evident by the lower binding affinity of p53TD to Con1, as manifested by split bands getting weaker as we go up in the gel pattern with the combined proteins.
The gel pattern with the Con1 target, and specifically the dimeric complex, is not a result of complex instability during the electrophoretic run. This can be determined from a time course of the reaction with the Con1 target, where we incubated this target with p53DBD in the regular conditions and loaded equal aliquots of the reaction at 10-min intervals. Supplementary Figure S3
shows that there is no accumulation of the dimeric species as a function of gel run time; on the contrary, the dimeric species became depleted as the complex migrated on the gel, due to gel instabilities. At the second shortest run time (from the right-hand-side of the gel), the dimeric species is 19 (1
)% of the total of the two bound bands, whereas at the longest run time it is 14 (2
)% of the total.
Changes in the CWWG motif leads to three orders-of-magnitude differences in the degree of cooperative binding
We have quantified the binding affinity in this study separately for each bound band using a two binding site model, shown for Con1, Con2 and Con3 in A–C. Such analysis assumes that the minimal binding unit for p53DBD is apparently a dimer. It is known that in solution p53DBD is monomeric (24
). However, the binding cooperativity for the monomer–dimer transition is faster than can be observed using these methods. More elaborate models, such as a four binding site model, resulted in significant worse fit between the experimental points and those of the model (data not shown). Since in gels with the Con2 and Con3 targets no bands of dimeric complexes were observed, we have included them in the analysis as having zero occupancy. This led to a significant improvement in the fit of the experimental data over that of using a one-site model (tetramer only), or using a two-site model without dimer occupancy included. However, analysis by two equations that do not include a term for Ka1
(dimer binding) resulted in the same tetramer Kd
The results of this analysis () showed significant changes between the targets in both the binding affinity and especially in the binding cooperativity. Con1 sequence showed the highest tetramer binding affinity in this series [64 (4
nM] and the lowest binding cooperativity [k12
), see ‘Material and Methods’ section for explanation on analysis method]. Con2 showed the lowest tetramer binding affinity [252 (19
nM] and the highest binding cooperativity (k12
000), and Con3 showed values between these two extremes [tetramer Kd
nM, and k12
~2500]. There is a 4-fold difference in binding affinity between the sites, and a 400-fold difference in the binding cooperativity. The binding affinity of p53DBD dimer to Con1 is 314 (14
nM. Cooperativity constants above ~1000 are not resolvable on EMSA gels (37
). They are indeed not observed in the gels of Con2 and Con3 and hence we added them in the analysis with zero occupancy in order to calculate the dimer binding affinity and cooperativity for these sites. The k12
values for Con2 and Con3 may therefore be only a rough estimate. However, the difference in k12
values among these sites, and between them and that of the Con1 target, is so large that it allows us to conclude that the changes in binding cooperativity between the sites are highly significant.
In all analyzed sequences, we observed a slight systematic underestimation of the bound bands, and a slight systematic overestimation of the free DNA bands, which is due to gel instabilities, at the 3–5 highest protein concentration. When there is only one bound species in the gel experiment (49
), or when we analyze the bound species together for overall affinity (12
), one can extend the bound complexes bands downwards to include such instabilities. However, here there is more than one bound species in the complex with Con1, and hence we delimited the boxes defining the bound complexes tightly around each band. This may also account for the difference in binding affinity of Con1 versus those studied by Kitayner et al
), where we performed an overall analysis, with boxes extended to the free DNA. Analysis for overall Kd
of the Con1 target led to a binding affinity of 20 (2
nM, similar to that of the GGG target of Kitayner et al
). However, only an analysis for separate binding of each species can demonstrate the vast differences in binding cooperativity between binding sites observed here. EMSA methods, carried out as described in this study, are known to yield robust and precise quantitative results (45
). Moreover, EMSA has been shown to be useful also for accurate quantitative determination of cooperativity in protein–DNA interactions, when analyzed as described by Senear and Brenowitz (37
), even when the analyzed gels are noisy.
Structural characterization of p53 sites as a function of the non-contacted step
There are many known instances in which DNA is deformed in protein–DNA complexes (54
). Hence, the different strength of binding and especially binding cooperativity may be due to different deformabilities of the studied targets (56
), especially as the targets differ in a region that is uncontacted by the protein in the protein/DNA complex. To address this issue we have measured the global structure and mechanical properties of decamers of Con1, Con2 and Con3 half-sites using the high-throughput DNA cyclization approach developed by Zhang and Crothers (32
). In this method, one measures the J
-factors of a series of DNA molecules, all containing the same test sequence (see ‘Materials and Methods’ section for details). The relationship between the experimental data and DNA structure is obtained by computer analysis, based on a model for the structural properties of the tested sequences. Specifically, two assays were performed – the phasing assay and the total-length assay (32
). In the first assay, the total-length is constant and the phasing between the test sequence and sequences containing curved DNA (A-tracts) is varied. Once the in-phase construct is determined, one varies the total length of the constructs, preserving the relative orientation of the two regions. The phasing assay is sensitive to DNA curvature and bending flexibility. The total-length assay is sensitive to DNA helical repeat and to bending and twist flexibility. Overall, 16 DNA constructs are used to determine four structural parameters for each sequence (twist angle, bend angle, torsional flexibility and bending flexibility).
shows the results of the phasing and total-length assays for Con1 to Con3. From the final structural parameters (), it can be observed that the significant difference between these sequences is only in their torsional flexibility. The Con1 decamer is one of the most torsionally flexible sequences determined to date, with 5.62 (0.09)° in twist fluctuations, which result in a torsional force constant of 1.432
erg·cm. Relative to the torsional flexibility of generic B-DNA measured by us [5.06 (0.06)°, ], both Con2 as well as Con3 half-sites are more torsionally rigid [4.64 (0.07)° and 4.71 (0.07)°, respectively]. To understand whether Con1 is unique in being torsionally flexible, we determined by cyclization kinetics the structural properties of three additional p53 consensus binding sites: GGG (also called Con4), GGA (Con5) and AGG (Con6), studied by Kitayner et al
). Supplementary Figure S4
shows the results of the phasing and total-length assay for these sequences. The final structural values () showed that all decamers with a CATG center are more torsionally flexible than sequences with a CAAG or CTAG center. The torsional force constant varies with the inverse square of the rms fluctuation angle. Looking at the values for the torsional force constant () we note that the Con4 target (1.395
erg·cm) is ~50% more flexible than either Con2 or Con3 (2.094 and 2.044
erg·cm, respectively). The twist flexibility of the most torsionally rigid DNA molecule, measured so far by cyclization kinetics, is 3.9 (0.3)° (56
), which amount to a force constant of 2.98
erg·cm. Thus, the whole range of sequence-dependent twist flexibility is ~2-fold. Previously estimation of the range of sequence-dependent torsional flexibility ranged from nonexistent (57
) to around 2-fold (58
). Thus, it seems that the ~2-fold range observed in DNA sequences studied so far by cyclization kinetics is about the entire range. Hence, the difference in the torsional force constant between p53 binding sites studied here is significant.
Figure 4. Cyclization kinetics of p53 REs as a function of the central CWWG motif. The J-factors for the DNA constructs as a function of either the phasing length (A–C), or the total DNA length (D–F), for Con1 (A and D), Con2 (B and E) and Con3 (more ...)
The high torsional flexibility of all CATG decamers is due to the combined flexibilities of the C-A (T-G) steps and not to that of the central A-T step. Using dimeric steps taken from B-DNA crystal structures (59
) or DNA structure in protein–DNA complexes (41
), Olson and co-workers note that these steps are the most torsionally flexible of all dinucleotide combinations, as inferred from the large dispersion values of these steps around their averaged values.
CTAG containing sequences were proposed to be exceptionally flexible (60
), on account of the known flexibility properties of T-A steps (61
). T-A steps are indeed anisotropically flexible, but their structure is also very context dependent, and thus sequences harboring T-A steps are not well represented by nearest-neighbor models (50
). We showed here that within the sequence context of the Con2 target, CTAG elements are torsionally rigid relative to generic B-DNA sequences.
The twist flexibility of the Con1–Con3 sequences and the binding affinity and binding cooperativity of these sequences show similar trends. The torsionally flexible Con1 target has the lowest binding cooperativity, the intermediate dimer species is long-lived and stable enough to show up as a separate entity on EMSA gels, and the affinity of the tetrameric complex is the highest of the series. The relatively more rigid Con2 and Con3 targets show only tetramer bands of lower relative affinity. We suggest that it is the flexible nature of the Con1 target that reduces its cooperativity of interaction and allows the formation of a long-lived and stable dimer intermediate, on the way to the functional tetramer species. The facile winding/unwinding motion of the CATG motif allow the stabilization of inter-subunit interactions within p53DBD, such that binding of only one dimer to DNA is stable, the cooperativity is thus low and the binding affinity is relatively high, for both the tetramer and the dimer (). This is not the case for the more torsionally constrained CAAG (Con3) and the CTAG (Con2) containing targets.
The significance of torsional flexibility in p53/DNA interactions can be clearly visualized by calculating the expected free energy difference of twisting p53 binding sites by the twist angle observed in crystal structures. In the GGG structure (Con4 in , the most torsionally flexible target), the difference between the A-T (24.1°) and the T-G (43.8°) steps is 19.7° in twist angle (12
). Similar differences in the center CATG core have been observed in other p53/DNA complexes with a CATG center (63
). The free energy difference for this amount of twist change is 2.43
erg or six times the thermal energy (kB
T) at 21°C (the temperature at which the torsional force constant was measured). For the Con2 target, the free energy difference for the same twist angle change is 6.64
erg, or nine times the thermal energy at 21°C. Thus, there is a 50% difference in the energetic cost of twisting these two targets.
It was previously suggested (17
) that DNA flexibility of p53 REs containing the CATG motif is an important factor in the enhanced binding affinity of such sites, but these discussions do not differentiate between axial (bending) flexibility and torsional (twist) flexibility. We showed that there is ‘no axial flexibility
’ intrinsic to the studied p53 targets, only ‘torsional flexibility
’. Nagaich et al
) observed, by gel studies, that a sequence identical to the AGG target (12
) is bent in solution in its free state. We did not observe any static bending, or any bendability of the sequences studies here and also not when the AGG (Con6, ) target was studied by cyclization kinetics. Our study is the first study to experimentally determine the global structure and mechanical properties of p53 REs by a sensitive and rigorous technique, carried out completely in solution, which does not rely on external calibrators and is based on a complete theoretical basis (66
In the recent crystal structure determination of a 20-bp duplex incorporating two contiguous decamers bound to p53DBD, each DNA half-site is only slightly bent, and the combined two half-sites are straight (67
). Bending of the magnitude displayed in this structure (as well as other crystal structures of p53DBD/DNA complexes, refs. 12,13,63,64,68) is accessible to generic B-DNA molecules that are not particularly axially flexible (the free energy of ~20° or lower overall bends /10
bp is below thermal energy at 25°C, ref. 54). Thus, the energetic penalty cost for formation of complexes with such bend magnitude is negligible. This is in line with our observations of an average axial flexibility in the three target sites examined here, regardless of their binding affinity. On the other hand, twist flexibility of p53 DNA targets may be functionally important. In the crystal structure determination of two contiguous decamers bound to p53DBD (67
), the base pairing at the central A-T doublet shows the Hoogsteen geometry. Further characterization, by the Shakked group (67
), of the mouse p53DBD tetramer covalently linked to a DNA duplex (64
), shows that a large fraction of the A-T central doublet is in the Hoogsteen geometry also in this structure. The ability of CATG containing DNA sequences, in complex with p53DBD, to adopt both the Watson–Crick as well as the Hoogsteen geometry is an indication of their large torsional flexibility, because the flip from Watson–Crick to Hoogsteen geometry and vice versa should involve winding and unwinding of the double helix.
Creating a torsional swivel in Con3 reduces its binding cooperativity and stabilizes a bound dimer
To further corroborate our suggestion that torsional flexibility is responsible for the difference in binding affinity and cooperativity between sites differing in the non-contacted step of the CWWG region, we introduced a torsional swivel into the Con3 target (nicked Con3, ) by designing it as an intramolecular dumbbell, containing two hairpin loops. The 5′ and 3′ ends meet at the middle of the 5′ half-site, and thus there is a nick in the 5′ half-site sequence between the C-A and A-G steps, which renders the nicked Con3 target highly torsionally flexible (as shown in other protein–DNA systems; refs. 31,32). The EMSA pattern for the nicked Con3 target and p53DBD () showed two bound bands, albeit less sharply defined than those of Con1. Quantitative analysis (D and ) was again carried out using a two binding site model. The analysis had now to be performed in terms of the macroscopic constants, since the two half-sites are not identical anymore. However, we can estimate the intrinsic binding constant of the nicked half-site (k2
) using the relationship Ka1
, assuming that we can take the intrinsic binding constant of the intact half-site (k1
) from measurements of intact Con3. Such analysis showed that the nicked Con3 half-site [600 (100)
nM, ] have ~20-fold higher affinity compared to the estimated Kd
of the dimer from the intact Con3 half-site. The cooperativity constant can be estimated from the expression of Ka2
and the known values for k1
, and it value [9.0 (0.6)] showed that the binding cooperativity to the nicked Con3 binding site is lower than that to the flexible Con1 site. DNA with a nick in the backbone (and with a 3′-end phosphate) was shown by Zhang and Crothers (32
) to have twist fluctuations of 6.8 (1.2)°, significantly more flexible than Con1. Such high twist flexibility allows the monomers within each dimer to re-adjust their relative conformation at low energetic cost, and thus they form stable and long-lived dimers, leading to low cooperativity of interaction of the nicked Con3 with p53DBD. This extends the trend observed with the Con1–Con3 series of inverse relationship between twist flexibility and cooperativity–high twist flexibility and low cooperativity of interaction and vice versa. However, here the trend does not include high affinity of tetramers of p53DBD binding to targets with high twist flexibility. The binding affinity of the tetramer complex to nicked Con3 was significantly reduced () relative to that of intact Con3, probably due to lose of contacts to the DNA backbone (12
Figure 5. Binding affinity measurements by EMSA of p53DBD binding to the Con3 target with a nick in the center of the 5′-half-site (as shown in ). This DNA target was synthesized as an intramolecular dumbbell construct with two hairpin loops. For (more ...)
Binding of p53DBD to p53 targets with spacers between half-sites
In the p53 consensus the two half-sites can be separated by up to 13
). More recent studies show, however, that spacer sequences of more than a few nucleotides decrease p53 responsiveness from these sites (11
), and attenuates p53 binding affinity (44
). We asked whether the differential effects of the non-contacted WW step shown above would differently affect binding, when spacer sequences are introduced into the Con1 versus the Con2 targets. For the Con1 target with spacers between half-sites the observed gel pattern (A and B) was again of two bound complexes, dimers and tetramers of p53DBD, as previously observed by Kitayner et al
). Comparing the binding pattern of Con1, Con1 and 2-bp spacer, and Con1 and 4-bp spacer side-by-side on the same gel (Supplementary Figure S5
) showed that the dimeric species migrated to the same distance on the gel in all complexes with these targets. The tetrameric species migrated to decreasing distances on the gel as a function of the spacer length. This may be due to differences in twisting of the DNA in the complexes. Quantitative analysis of the binding affinity of p53DBD to these sites (E and F and ) showed that the dimeric species bound with slightly reduced Kd
to that observed in the Con1 target [442 (20
nM and 581 (46
nM for the insertion of 2- and 4-bp spacer respectively, ]. Significant reduction in binding affinity relative to that in Con1 was observed for the tetrameric species [435 (32
nM and 1.02 (0.06)
µM for the sites with 2- and 4-bp spacers respectively, ], corresponding to reduced protein–protein interactions in the inter-dimer interface, in comparison to protein–protein contacts in the complex with contiguous DNA site (67
). The cooperativity of interaction is reduced from that observed for Con1 to 1.1(1
) with 2-bp spacer and 0.34 (0.06) with 4-bp spacer (). Altogether, the cooperativity differences between p53 target sites spanned five orders of magnitude, as a function of binding site sequence and spacers. No significant binding, of either binding stoichiometry, can be observed to the Con2 targets with spacer sequences (C and D). Analysis (G and H and ) showed that the binding affinity for both species is in the micromolar range.
Binding affinity measurements by EMSA of p53DBD binding to REs with spacers between half-sites. (A) Con1 with 2-bp spacer. (B) Con1 with 4-bp spacer. (C) Con2 with 2-bp spacer. (D) con2 with 4-bp spacer. For details, see .
Statistical analysis of validated p53 half-sites grouped by the central CNNG motif
We have measured in this study the flexibility of six defined p53 consensus targets, with variations in the CWWG motif at their center, and noted a significant difference in the observed torsional flexibility of the A-T containing targets from that observed in the T-A or the A-A targets. Other central dinucleotide steps are found in natural p53 REs (within CNNG motifs), and they are imbedded within different flanking contexts, which have a consensus of RRR (and YYY), but from which most p53 REs deviate, in at least one position (42
). We wanted to assess the centrality of the structural properties of the core CNNG motif in determining the overall intrinsic flexibility of p53 RE half-sites (that is along the entire decameric half-site). Thus, we asked whether we can group all available p53 RE half-sites, based on the identity of the central tetranucleotide, and whether such clustering will result in groups that are significantly distinct from each other, based on their structural parameters. In order to carry out such analysis, we assumed that we can represent DNA sequences based on nearest-neighbor interactions only, so that we can carry out calculations on the structural deformability of DNA sequences from knowledge of the deformability of all DNA doublets. Olson et al
) have studied the sequence-dependent deformability of DNA doublets, based on their conformation in protein–DNA complexes. This data set was the only available dinucleotide properties dataset that when we used it to calculate the torsional flexibility of the six p53 binding sites for which we have cyclization kinetics data, we got a good-enough correspondence between the dispersion values of dinucleotide steps around their mean value and our experimental measurements (ρ
0.04) to suggest that these dispersion values can be used as a measure for the intrinsic sequence-dependent flexibility of DNA base-pair steps.
DNA structural parameters that show the largest variability, as a function of base-pair step identity, are twist, roll and slide (41
). We therefore calculated the mean twist, roll and slide flexibility for each known p53 RE half-site (42
), using the dispersion values from Olson et al
), and then calculated the mean of the mean for sequences grouped by the central tetranucleotide. We included in this analysis only p53 REs that have decameric half-sites and that contain a central CNNG core. The results (Supplementary Table S1
) showed that sequences with a CATG center have the highest torsional flexibility, slide flexibility and roll flexibility, whereas sequences with a CTAG center have the lowest flexibility in these parameters, relative to CATG or CAAG, even though now the sequences have various sequence motifs flanking a specific central tetranucleotide core. We carried out statistical tests to assess the significance of these results (Supplementary Table S2
), which showed that the differences between CATG and either CAAG or CTAG groups are highly significant (P
-values are 4.0E-10 and lower), in either rotational twist flexibility or translational slide flexibility. Overall, the differences between the groups with respect to changes in the roll parameter are of lower significance. Many other significant differences can be noted, especially between CATG and other groups, or CTAG and other groups, because these two groups are the two extreme ends of values of flexibility parameters. Even if we multiply the P
-values by 45, to account for multiple comparisons (15 comparisons and by three parameters, ref. 43) many significant differences remain, especially between CATG and other groups, and CTAG and other groups. Changes in twist, slide and roll are not independent of each other (41
); however, as the differences between the groups are so significant, there is no need to carry out a multivariate analysis to test for significant clustering patterns using twist, slide and roll dispersion together.
Function from p53 half-sites follows their binding and structural characteristics
It is now established that p53 can function also from p53 half-sites. Menendez et al
) showed that a p53 consensus half-site generated by a C to T SNP in the flt-1 promoter can support ~25% of the transactivation level of the p21-5′ RE. Combined with a nearby estrogen receptor RE, and upon treatment with estradiol, the transactivation level increases 4-fold, to that from the potent p21-5′ RE (73
). That p53 can transactivate from p53 half-sites was also established from half-site RE found in the RAP80 promoter (74
), where only slight reduction is observed relative to the full RE (the full RE contains a 4-bp spacer). These results expand the universe of potential p53 RE and with it the ability to fine tune p53 regulated genes. A clear trend can be established between the identity of the CWWG motif and transactivation from p53 half-sites (20
). A sequence identical to Con1HS (with a CATG center) showed the highest transactivation level of all CWWG half-site variants, followed by Con3HS (with CAAG, 5-fold reduction in transcription level), whereas Con2HS (with a CTAG center) hardly showed any transactivation ability (20
). This follows our binding affinity measurements as well as the torsional flexibility of the CWWG sites studied here. Based on our results we interpret these observations as resulting from the inability of p53 to bind CTAG containing half-sites, which is due to the relative torsional rigidity of such sequences.
Cooperative binding by p53DBD—implications for functional binding by full-length p53 and by p53 isoforms
Here, we studied the binding characteristics of p53DBD to several consensus target sites. This is not the functional form of p53 in vivo
. However, it is now known that the p53
gene encodes at least ten different p53 protein isoforms as a result of alternative splicing (75–78
). Some variants lack part of the N-terminal region (the TA domain), whereas other variants lack the parts of the tetramerization domain and/or the extreme C-terminal regulatory domain (78
). It has been suggested that p53 isoforms may modulate wt p53 (the canonical form of p53) activity by modulating DNA binding, by the formation of hetero-oligomers, and/or by sequestering p53 in the cytoplasm. Alternatively, p53 isoforms may have autonomous specialized functions (78
). This may be isoform dependent and/or cell-type specific (77
). Furthermore, p53 isoforms arise most commonly because of splice-site mutations, which may lead to exon skipping, or the creation of new splice sites (79
). Although at this stage the observations on p53 isoforms, in normal and cancerous cells, are sometimes conflicting (discussed in refs. 78 and 81–83
), advancing our detailed understanding of the ability of various p53 truncation constructs to bind various p53 target sites is important for cancer diagnosis and treatment.
We showed in this study that there are five order of magnitude changes of cooperative binding of p53DBD to specific targets as a function of changes in consensus sequences. Whenever the cooperativity constant is above ~1000 no intermediate species are observed (37
). Indeed, for complexes of p53DBD with the Con2 and Con3 targets no dimer bands are observed. Nonetheless, there is a significant change in cooperative interaction between these two sites. The binding of full-length p53 to DNA was suggested to be as cooperative (or more) than the binding of p53DBD to DNA (24
). We suggest that binding of full-length p53 to DNA may show similar cooperativity changes as a function of DNA base sequence, even though it may bind to it REs only as a tetramer, because of higher cooperativity of interaction. Further studies will provide information on the effect of domains outside the DBD on sequence-dependent binding cooperativity of p53 to its REs.