From the Lineberger Comprehensive Cancer Center and the Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599-7295
Received for publication, July 28, 2000, and in revised form, December 18, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The tumor suppressor protein p53 modulates
cellular response to DNA damage by a variety of mechanisms that may
include direct recognition of some forms of primary DNA damage. Linear
49-base pair duplex DNAs were constructed containing all possible
single-base mismatches as well as a 3-cytosine bulge. Filter binding
and gel retardation assays revealed that the affinity of p53 for a
number of these lesions was equal to or greater than that of the human mismatch repair complex, hMSH2-hMSH6, under the same binding
conditions. However, other mismatches including G/T, which is bound
strongly by hMSH2-hMSH6, were poorly recognized by p53. The general
order of affinity of p53 was greatest for a 3-cytosine bulge followed by A/G and C/C mismatches, then C/T and G/T mismatches, and finally all
the other mismatches.
Loss or alteration of p53 function occurs in about half of all
human cancers, and the investigation of its role in prevention of tumor
development has placed it in a key position within a complex network of
interactions (reviewed in Ref. 1 and 2). Among the known functions of
p53, one of the best characterized is the transcriptional regulation of
certain genes in response to conditions threatening the integrity of
the cell (reviewed in Ref. 3-5). DNA damage caused by UV and X
irradiation (6, 7), hypoxia (8), depletion of ribonucleoside pools (9), the expression of several oncogenes (Ref. 10 and references therein),
and other genotoxic conditions all lead to the arrest of cellular
growth or apoptosis (11) through p53-mediated pathways (12). These
events appear to be triggered by an activation and/or accumulation of
p53 in the cell, but the detailed mechanisms are not fully understood.
Growing evidence has revealed the importance of post-translational
modifications such as phosphorylation and acetylation of p53 in this
process (13, 14). Modulation of p53 activity in most cases is
influenced by changes in its DNA binding properties, including
alterations in the regulation of binding by the C-terminal domain
(reviewed in Ref. 15). Additionally, p53 binds its transcriptional
response element as a tetramer, and disruption of this conformation
leads to a decrease in binding activity (16).
p53 function in the cell is not limited to transactivation. Studies
in vivo and in vitro have shown that p53
associates with proteins involved in DNA repair (such as XPB, XPD (17),
and WRN (18)), in recombination (hRad51 (19)), and in replication (DNA
polymerase Although it is generally assumed that p53 acts via mediator proteins in
response to primary damage in DNA, there is growing evidence that p53
can bind directly to sites of DNA lesions. p53 can recognize and bind
single-stranded (ss)1 DNA and
double-stranded (ds) DNA ends (24) in a sequence-independent manner.
Both types of breaks represent intermediates of DNA damage, repair, and
recombination. Additionally, p53 binds tightly in vitro to
recombinational intermediates such as Holliday junctions and in doing
so enhances their resolution by junction cleaving enzymes (25). p53
also binds three-stranded DNA structures (25, 26). Finally, we
demonstrated that p53 and its C-terminal fragment form stable complexes
at sites of extra base bulges (insertion/deletion mismatches) in
vitro (27). When two similar substrates that contain some
nonhomologies undergo recombination, clusters of extra base bulges and
single-base mismatches are left behind, and their presence must be
signaled for repair. Extra base bulges and single-base mismatches may
also occur as a result of replication errors or repair of DNA damage
induced by irradiation or carcinogens. For example, the presence of
lesions such as thymine dimers could result in the insertion of
nucleotides opposite the noninformational site during translesion
synthesis, thus leaving behind mispairs and bulges. Collectively, these
data suggest that a direct interaction of p53 with such DNA lesions may
be a relevant upstream event in the biological function of this
protein. However, the role of DNA damage intermediates in activating
p53 following DNA damage is not fully understood, nor is the full
spectrum of lesions to which p53 may respond known.
Currently, two models for how p53 binding to sites of damage might
alter events in the cell have been suggested. In one, p53 binding to
lesions causes transcriptional activation of a cascade of genes leading
to growth arrest or programmed cell death (28), but the mechanism of
p53 transactivation of genes located at a great distance when tethered
to sites of DNA damage was unclear (29). Recently, it has been shown
that once bound to an extra base bulge in DNA, p53 can dissociate in
the presence of ATP (30). It is possible that the protein dissociates
in an altered form (for example, a structural alteration or a
modification such as phosphorylation) and provides the appropriate
signals at a distance from damage sites.
The second model proposes a more direct participation of p53 in the DNA
repair and recombination pathways (26, 29). The population of p53 in
cells may not be homogeneous and may contain a mixture of molecules
with different post-translational modifications (29). Thus, some forms
of p53 could preferentially bind to response elements, whereas others
may show a higher affinity for lesions in DNA. p53 molecules that bind
to DNA lesions could, in turn, signal their presence to loci containing
p53 response elements as well as recruit one or more proteins with
which it interacts. It is also possible that in vivo, p53
exists primarily within different multiprotein complexes. Some of these
could be specific for the recognition and repair of DNA damage and
might include members of the mismatch repair protein family.
Given the growing evidence that p53 is a potential damage recognition
protein, it has become important to survey a large number of lesions in
DNA to determine the extent to which each is bound by p53. Some lesions
may directly induce p53 binding, whereas other lesions may be
recognized by different proteins that act as a signal for the p53
response. Determination of the spectrum of lesions to which p53 does or
does not bind directly is thus critical to further development of
models that explain the role of this protein in the cellular response
to DNA damage. Although binding of p53 to extra base bulges has been
examined, its ability to directly recognize single-base mismatches has
not. Dudenhoffer et al. (26) designed DNA substrates
consisting of a recombinational intermediate with single-base
mismatches and observed p53 binding to these structures when they
contained A/G but not G/T mismatches. However, the complexity of these
structures made it difficult to determine the influence of the mismatch
component alone. Therefore, analysis of p53 binding to single-base
mismatches in otherwise simple linear DNA is crucial. To address this
need, we generated duplex DNA substrates containing all possible
single-base mismatches and have examined their interactions with p53.
Single-base mismatches and extra base bulges are repaired by the
mismatch repair system. A number of these mismatches are recognized by
the Escherichia coli mismatch repair protein, MutS and its
eukaryotic homologs (MSH2, MSH3, and MSH6) with differing affinities
found for the various mismatches in vitro (Ref. 31 and
references therein). p53 and the mismatch repair proteins share many
similar functions such as the ability to recognize extra base bulges
(27, 32, 33) and Holliday junctions in vitro (25, 34) as
well as inhibition of DNA recombination upon encountering mismatches
in vivo and in vitro (26, 35, 36). To place the
in vitro p53 binding studies within the context of repair
events in the cell, the affinity of p53 and hMSH2-hMSH6 for the various
mismatches was compared.
DNA Substrates and Proteins--
Double-stranded DNA substrates
(49 bp) were made by annealing complementary single strand
oligonucleotides (high pressure liquid chromatography purified
from Life Technologies, Inc.). The duplex DNA substrates were
end-labeled with [ Filter Binding Assays--
For filter binding studies, reactions
(20 µl) were carried out in a DNA binding buffer containing 20 mM Hepes (pH 7.5), 10% glycerol, 50 mM KCl,
0.1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride) for 20 min at room temperature. The
amount of DNA substrate, competitor DNA, and protein are specified in
the figure legends. The samples were applied to presoaked
nitrocellulose protein-binding filters (Protran; Schleicher & Schuell)
at a flow rate of 1 ml/min by vacuum suction. Each filter was washed
twice with the DNA binding buffer and air-dried for 30 min.
Radioactivity was measured by scintillation counting. Binding of the
DNA alone to the filters in the reaction mixtures (protein-free
background binding) was less than 5%. The amount of p53-bound DNA was
calculated as the ratio of the cpm/filter (minus the protein free
background) to the total cpm of the reaction mixture.
Gel Retardation Assays--
The standard assay for p53 and
hMSH2-hMSH6 binding was performed in a buffer optimized for hMSH2-hMSH6
studies (39) containing 25 mM Hepes (pH 7.5), 15%
glycerol, 50 mM NaCl, 1 mM dithiothreitol, 0.01 mM EDTA in a 20-µl reaction volume. The concentration of DNA substrates, protein, and unlabeled competitor DNA are indicated in
the figure legends. Unlabeled lesion-free dsDNA (Fig. 1) was used as
nonspecific competitor DNA. The reactions were incubated at room
temperature for 20 min unless otherwise indicated. Upon completion, the
samples were separated on 5% polyacrylamide gels in 0.5× TBE buffer
(44.5 mM Tris, 44.5 mM boric acid, 2 mM EDTA) at 200 V at 4 °C for 2 h. Gels were dried
and quantified using a Storm 840 PhosphorImager (Molecular Dynamics).
Filter Binding Analysis Reveals p53 Binding to Some Mismatches in
Duplex DNA--
The affinity of p53 for various forms of damage in
linear duplex DNA typical of those generated during replication or
recombination needs to be determined to more fully understand how p53
responds to genetic insult of this form. To carry out these
experiments, we designed a series of linear 49-bp DNA templates (Fig.
1). The lesion-free DNA contains no
damage and was based on a template described previously (27). Variants
were constructed containing all mismatches located centrally: A/C, A/G,
C/T, G/T, A/A, C/C, G/G, or T/T. Each duplex DNA template contained
three lesions spaced by 2-4 bp. This design was based on studies of
Alani et al. (Ref. 40 and references within) and Lee
et al. (27) showing that the presence of the triple lesion
significantly amplified the binding signal in gel retardation and
filter binding assays. Further, clusters of mismatches are frequently
left behind following recombination between two similar but not
identical DNAs. As a positive control for p53 lesion binding, a
template containing a single 3-cytosine bulge was synthesized. Two
additional controls for testing the DNA binding properties of p53 were
also designed. The first template contained the full 20-bp human GADD45
p53 response element (41) at the center, and the second contained the
same response element with 4 nucleotides shown to be particularly
important for p53 binding substituted with other bases. Because ssDNA
alters p53 binding (28, 42), the duplex templates were first incubated with benzoylated naphtoylated DEAE-cellulose and then purified from
nondenaturing 15% polyacrylamide gels to remove any contaminating ssDNA. For all the studies below, a single buffer previously optimized by Gradia et al. (39) for hMSH2-hMSH6 binding studies was
employed. Comparative studies showed no significant difference (data
not shown) when this buffer was used for p53 as contrasted to buffers containing magnesium (43), whose inclusion raised the potential complication of the activation of the putative exonuclease activity in
the p53 (44).
Filter binding assays were employed to evaluate p53 binding to these
substrates. The human p53 used here was produced in SF9 insect cells,
and analysis of this protein indicated that there was at least one
phosphorylated site.2
End-labeled DNA (1 nM) was incubated with p53 (15 nM of tetramers) for 20 min in DNA binding buffer
("Experimental Procedures") at room temperature in the presence of
a 15-fold excess of unlabeled nonspecific competitor DNA. The results
(Fig. 2) showed that p53 exhibits
different affinities for substrates with different mismatches. p53
showed low binding to the negative controls: 4.2% of the lesion free
and 3.8% of modified GADD45 templates were protein bound. The
3-cytosine bulge substrate, which serves as a positive control for
lesions, showed the high binding (14% of the probe was bound by p53).
Similarly, p53 bound extremely well to the full GADD45 response element
(38% of binding). For the DNA containing A/G mismatches, 5.8% of the
probe was bound by p53, whereas binding to DNA with C/C was 7.1%.
Binding of p53 to DNA containing the other mismatches was lower than
4%. When higher concentrations of p53 were used, a small increase in
binding was observed (except for C/C); however, the relative order of
affinity remained the same: 3-cytosine bulge > C/C > A/G > wild type > A/C > G/T (Table I). It should be noted that a titration
of p53 on these probes in the absence of nonspecific competitor showed
higher levels of binding (data not shown).
Gel Retardation Analysis Confirms p53 Binding to Some Mismatch
Containing DNAs--
Although filter binding assays provide a
comparison of the total amount of bound DNA with the different
substrates, this method does not allow direct visualization of
protein-DNA complexes. A number of authors have documented nonspecific
binding of p53 to DNA (Refs. 24 and 45; reviewed in Ref. 29). To
evaluate specific mismatch-dependent interactions of p53 to
these substrates, gel retardation assays were performed.
The multi-staged process of substrate purification and the necessity of
using different single-stranded oligonucleotides to produce the
double-stranded substrates excluded the possibility of having both the
same amount of radioactivity and the same concentration of DNA for the
different substrates. Therefore, binding of p53 to mismatches was
tested in two different ways. First, probes were added to the reactions
so that they contained the same amount of cpm for each substrate. As
mentioned above, this required the addition of different concentrations
of DNA. Therefore, different amounts of p53 were added to each probe to
ensure a 1:10 molar ratio of DNA to protein. The DNA substrates and p53
were incubated for 20 min at room temperature in the presence of a
20-fold excess of nonspecific unlabeled competitor DNA followed by gel
retardation assays ("Experimental Procedures"). p53 formed
complexes with some of the mismatched substrates as seen by the
retarded mobility of the probes, and complex formation varied depending
on the type of the mismatch. As shown in Fig.
3, binding was greatest for DNA with a
3-cytosine bulge (Fig. 3A, lane13) and
progressively less for the modified GADD45 sequence (lane
14), followed by the C/C (lane 10) and C/T containing
substrates (lane 11). Finally, binding to the G/T
(lane 12) and A/C (lane 9) containing DNAs was
weak but greater than the lesion-free DNA, which showed no binding
(lane 8). p53 also showed modest binding to the modified GADD45 probe. Quantitative phosphorimaging analysis shows that p53
binding to the 3-cytosine bulge was 48-fold greater than lesion-free DNA, whereas binding to DNA with the modified GADD45 sequence was
11-fold higher than lesion-free DNA (Fig. 3B). The fold
increase in binding for the C/C, C/T and G/T containing substrates as
compared with the lesion-free DNA was 8.7, 6.0, and 4.6, respectively. The A/C containing substrate behaved similar to the lesion-free DNA.
Using a second approach, experiments were set up such that the
concentration of DNA in each of the reactions was the same, but because
of the reasons described above, the amount of radioactivity for each
substrate was not the same. Each reaction contained 3 nM
DNA substrate, 40 nM p53 (tetramer), and a 15-fold excess
of unlabeled dsDNA (control reactions without p53 are not shown). Gel
retardation assay (Fig. 4) shows a series
of distinct slow migrating bands corresponding to the position of the
p53-DNA complex (Fig. 4A, lane 11,
arrows a, a', and a"). In addition,
there was a smear of faster migrating material (Fig. 4A,
bracket c) that probably represents nonspecific complexes of
monomeric p53 with the substrates. These gel retardation assays were
carried out multiple times (n = 3), and protein-DNA
complexes were quantified. The level of p53 binding to mismatches was
compared with the binding to the wild type substrate. The results
showing the highest binding were seen with the substrate containing a
C/C mispair, followed by A/G, and finally A/C and T/T. DNA containing
C/T, G/T, A/A, and G/G mismatches showed binding that was lower than
the wild type DNA. As expected, the highest binding was seen with the
3-cytosine bulge.
Over the course of these experiments several different preparations of
p53 were utilized. Even though the differences between binding to the
various mismatches were not great in some cases, the hierarchy of
binding to the different substrates was the same among the different
p53 and DNA preparations.
p53 Binding to Lesion-containing DNA Is
Concentration-dependent--
To ensure that the relative
affinities of p53 for the different substrates described above were not
due to fortuitous choices of DNA or p53 concentrations, binding of the
protein in the gel retardation assays was followed as a function of DNA
concentration. The concentration of DNA substrates was increased from
0.75 to 7.7 nM, whereas the concentration of p53 was kept
constant at 12 nM. Fig. 5
shows representative binding curves from some of the mismatches tested.
In all cases, the lesion-free substrate was included as a control.
Binding was modeled by PRISM software (GraphPad Inc.), assuming only
one binding site (a cluster of mismatches) per DNA template. p53
binding to DNA containing the GADD45 site increased by 5-fold between
the lowest and highest concentration of substrate tested (data not
shown). At equal concentration of DNA ligands, the amount of bound DNA
was higher for the substrates containing the A/G (Fig. 5A),
the 3-cytosine bulge (Fig. 5B), and C/C (data not shown)
lesions than for the lesion-free DNA. Substrates containing A/C, C/T,
G/G, and T/T mismatches showed binding curves similar to the
lesion-free DNA (curves for A/C and C/T shown in Fig. 5A).
On the other hand, substrates containing G/T and A/A mismatches showed
binding curves lower than lesion-free DNA (Fig. 5B).
Although we used a mathematical model describing one-site binding to
generate the binding curves, this model does not completely fit the
experimental conditions. Specific p53-DNA complexes could be affected
by factors such as the presence of DNA ends, which could provide
additional sites that might compete for binding. Thus, efforts to
calculate association/dissociation constants for p53 based on one-site
binding models may not be applicable in this case.
A similar series of gel retardation assays were carried out in which
the DNA concentration was held constant while the p53 concentration was
varied. The data shown in Fig. 6 indicate
that the rate of increase in p53 binding for the four substrates
examined was different. For example, changing the p53 concentration
from 12 to 40 nM led to a 4.3-fold increase in binding to
the lesion-free substrate and a 3.2-fold increase in binding of the
substrate containing the A/C mismatch. In contrast, 8.9- and 7.2-fold
increases were observed for the A/G and C/C substrates, respectively.
The binding curves appear to be bi-phasic with little increase between 3 and 20 nM followed by a marked rise when the
concentration was increased from 20 to 40 nM. Indeed, at 28 and 40 nM p53, very significant differences were seen in
binding to the A/C and lesion-free substrates in contrast to the
substrate containing the C/C mismatch. Consistent with the results
above, the affinity of p53 for the lesion-free DNA was not the lowest
observed; rather the substrate with A/C mismatches bound p53 even less
effectively. Such effects have been observed in other studies (see
"Discussion").
The Stability of p53-DNA Complexes Varies Depending on the Lesion
in the DNA Substrate--
The stability of the p53-DNA complexes can
be probed by competition analyses using increasing concentrations of
nonspecific competitor DNA. Here, equal amounts of radiolabeled
substrates were incubated with p53 for 20 min at room temperature,
followed by addition of increasing amounts of unlabeled competitor DNA for a further 15 min. As shown in Fig. 7,
when a 200 fold excess (500 ng) of competitor DNA was added, p53
binding to the GADD45 and 3-cytosine bulge containing substrates was
reduced by less than 2-fold (from 83 to 58% and from 84 to 45% of the
DNA bound, respectively). In contrast, under the same conditions
p53-DNA complexes were reduced to ~3% when competitor was added to
DNAs containing A/C or G/T mismatches or the lesion-free DNA, arguing that these complexes are much less stable. When p53 was incubated with
DNA substrates containing A/G or C/C mismatches followed by addition of
a 200-fold excess of competitor, 8 and 13% of the DNA remained in
complex with p53. Thus, the hierarchy of complex stability was as
follows: the highest for the GADD45 and 3-cytosine bulge containing
substrates, then DNAs with A/G or C/C mismatches, and lowest for A/C
and G/T containing substrates, which is in agreement with the results
above.
p53 Has a Higher Affinity for Some Mismatches than Does
hMSH2-hMSH6--
The work above and previous studies (27, 26) provide
evidence that p53 can directly recognize certain single-base mismatches and bulges in DNA. hMSH2-hMSH6 is a heterodimeric protein involved in
the primary recognition steps of mismatch repair in eukaryotic cells
(46, 47). The observations made here would be of particular importance
were it found that the relative affinities of p53 and hMSH2-hMSH6 under
similar experimental conditions were comparable for at least some of
the substrates examined. The incubation buffer used here and in the
experiments above for p53 is one previously optimized for hMSH2-hMSH6
binding studies by Gradia et al. (39). The mismatched
substrates were incubated with p53 or hMSH2-hMSH6 for 20 min at room
temperature at the same DNA:protein molar ratio (1 DNA molecule/35 p53
tetramers or hMSH2-hMSH6 heterodimers) in the presence of nonspecific
unlabeled competitor DNA. Using equal DNA concentrations of each probe,
the proteins were incubated under the same binding conditions
("Experimental Procedures"). The affinity of hMSH2-hMSH6 for the
3-cytosine bulge was similar to that of p53 (Fig. 8,
A, compare lane
13 with lane 6, and B). hMSH2-hMSH6
recognizes G/T containing substrates more strongly than p53 (Fig. 8,
A, lanes 5 and 12, and B)
as expected because hMSH2-hMSH6 is known to have the strongest affinity
for G/T mismatches as compared with all other mismatches (summarized in
Ref. 31). Indeed, as seen in Fig. 8, hMSH2-hMSH6 showed much lower
binding to the other mismatches. Among the other mismatches,
hMSH2-hMSH6 only showed higher binding to the A/C mismatch as compared
with p53. All the remaining mismatches tested were bound with higher affinity by p53 than hMSH2-hMSH6 (Fig. 8B).
Determination of those lesions and unusual structures in DNA that
generate signals for p53 binding is central to further understanding the response of this protein to cellular DNA damage. This is of particular importance because we previously showed that p53 strongly recognizes Holliday junctions and insertion/deletion mismatches in the
form of extra base bulges (27, 25). In this study, we examined the
ability of purified human p53 protein to bind to single-base mismatches
in linear DNA. Additionally, the affinity of p53 for the various
mismatches was compared with the human mismatch repair protein,
hMSH2-hMSH6, under the same conditions.
The mismatched substrates we used contain a cluster of three mismatches
separated by 2-4 bp. Our rationale for using a cluster of mismatches
was severalfold. First, such clusters were found by Alani et
al. (40) to provide stronger signals for yMSH2 binding than
single-base mismatches. Second, we assumed that if no binding was seen
to a 3-mismatch cluster, then there would be little reason to probe
binding to a single mismatch alone. Third, such clusters are commonly
found following homologous recombination through similar but not
identical DNAs, making them an inherently interesting lesion in DNA. It
is possible that for some of the mismatches, the close proximity of
three mismatches could destabilize the helix and create a transient
single stranded bubble to which p53 would bind. However, were this the
case, these bubbles should create strong binding sites for hMSH2-hMSH6
and just the opposite was observed; the mismatches bound best by p53
were not recognized by hMSH2-hMSH6.
Filter binding and gel retardation assays were carried out in several
different ways to examine the affinity of p53 for linear DNAs
containing single-base mismatches. Although the results varied slightly
among the approaches, they were in good agreement and revealed the same
pattern of p53 binding to the different substrates. As a positive
control and to generate a link with the hMSH2-hMSH6 results, binding
was compared with a substrate containing a single 3-cytosine bulge. p53
and hMSH2-hMSH6 showed strong and roughly equal affinities for this
lesion as seen by gel retardation and filter binding assays and matched
previous reports (summarized in Ref. 31). In all of the assays, p53
reproducibly showed the highest affinity for DNAs containing A/G and
C/C mismatches, and this was always higher than binding to the
lesion-free DNA. Competitor titration (Fig. 7) also showed that the p53
complexes formed on substrates with A/G and C/C mismatches were more
stable than ones formed on templates containing other mismatches or
with the lesion-free DNA. Two of the substrates containing G/T and C/T
mismatches did not show significant p53 binding above the lesion-free
DNA level (but were never lower). It is interesting to note that
Dudenhoffer et al. (26) demonstrated that p53 had a higher
affinity for a recombinational intermediate substrate containing an A/G
mismatch than ones lacking a mismatch or with A/C and G/T mismatches.
Although they did not examine all possible mismatches as described here and their substrates were relatively complex, our results are nonetheless in good agreement with their findings.
Substrates with A/C, A/A, G/G, and T/T mismatches showed binding that
generally did not exceed that of the lesion-free DNA, and in several
experiments, these substrates showed levels of binding that were
reproducibly lower than the control DNA. The observation that certain
base/base mismatches present a particular sequence context can lead to
reduced protein binding was noted previously in our studies with the
recA protein (48). Binding of recA to linear duplexes containing C/C,
G/G, T/T, and C/T mismatches was depressed relative to the lesion-free
DNA (48). Further, Dudenhoffer et al. (26) noted that the
binding of p53 to complex recombinational intermediates containing
mismatches was lower when the DNA contained a C/T mismatch in contrast
to homoduplex DNA. The explanation remains unclear but points to the
importance of sequence context in the recognition of both
transcriptional activator sites as well as lesions.
The potential biological significance of p53 binding to the different
mismatches was examined by comparing its affinity for these lesions to
that of the human mismatch repair complex, hMSH2-hMSH6 (Fig. 8). The
substrate containing the 3-cytosine bulge was used as a positive
control for both proteins, and our results show that the affinity of
p53 for this lesion equals that of hMSH2-hMSH6. In agreement with
previous reports (31), hMSH2-hMSH6 binds G/T mismatches with a markedly
higher affinity than any other single-base mismatch, and this was found
to be equal to its affinity for the 3-cytosine bulge. All other
single-base mismatches are recognized by hMSH2-hMSH6 within a range
that is severalfold lower than the G/T mismatch (Fig. 8). Nevertheless,
these mismatches are still signaled for repair by the mismatch repair
proteins. Therefore, the lower binding of hMSH2-hMSH6 to the mismatches
must be within a biologically significant range. p53 binding to all the
single-base mismatches fell in the lower range exhibited by
hMSH2-hMSH6. However, p53 had higher affinity for several mismatches
than hMHS2-hMSH6. These data suggest that the binding of p53 for
several of the mismatches (A/G and C/C) is potentially biologically
relevant because they lie within the range of hMSH2-hMSH6 binding.
Clearly, in vivo, these affinities will be altered by
modifications to the proteins (below) and by the binding of other
proteins. Nonetheless, the affinity of p53 alone for these lesions, as
measured here, provides a needed base line for understanding whether or
not these proteins have significant potential to respond to such damage in vivo.
The DNA binding properties of p53 are influenced by post-translational
modifications such as phosphorylation and acetylation (12). The p53 and
hMSH2-hMSH6 proteins we used were generated in insect cells, a common
source of p53 or hMSH2-hMSH6, and both have been used in previous
published work on mismatch and Holliday junction recognition (27, 25,
39). It has been documented in several recent papers that the
post-translational status of p53 may influence its binding properties
(13, 14). Analysis of the phosphorylation state of the baculovirus
expressed p53 used here by two-dimensional gel
electrophoresis2 indicates that it has at least one
phosphorylated site. It was essential that we first carry out studies
using proteins purified from insect cells as we and others have done
before to establish a foundation of understanding how p53 recognizes
different lesions in DNA and to compare it with similar studies with
hMSH2-hMSH6. With this study completed, it will be important in the
future to determine how individual site-specific modifications of p53 alters its affinity for different lesions in DNA. Additionally, the
effect of other proteins on p53 binding to the various lesions also
needs to be examined.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(20) and RPA (21)), thus pointing to the direct
participation of p53 in these processes. This hypothesis has been
strengthened by several studies in vivo suggesting an involvement of p53 in the control of recombination and repair (7, 22,
23).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP using T4 polynucleotide
kinase (New England Biolabs Inc.). Benzoylated naphtoylated
DEAE-cellulose (Sigma) was used to remove any contaminating ssDNA (37).
The substrates were desalted using Sephadex G50 spin columns (Amersham
Pharmacia Biotech) and then purified on 15% nondenaturing
polyacrylamide gels. Human p53 protein was overexpressed in SF9 cells
using a vector provided by Dr. Arnold Levine and purified as described
previously (38). The hMSH2-hMSH6 heterodimer was purified as described
previously (39) using a vector provided by Dr. Richard Fishel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (58K):
[in a new window]
Fig. 1.
DNA substrates. A 49-bp sequence free of
any p53 binding motifs (WT) was used as a template for the
DNA duplexes containing A/C, A/G, C/T, G/T, A/A, C/C, G/G, and T/T
mismatches and one with a single extra helical 3-cytosine bulge
(BC). The GADD45 and modGADD45 DNAs are 49-bp homoduplexes
containing the human GADD45 response element and a modified variation
(with the changes underlined).
View larger version (23K):
[in a new window]
Fig. 2.
Mismatch binding activity of p53 determined
by filter binding assays. p53 (15 nM) was incubated
with 32P-labeled DNA templates (1 nM)
containing A/C, A/G, C/T, G/T, A/A, C/C, G/G, and T/T mismatches, a
3-cytosine bulge, and the GADD45 or modGADD45 response elements in
presence of unlabeled competitor dsDNA (15 nM). Filter
binding assays and quantification of DNA-protein complexes were
performed as described under "Experimental Procedures."
WT, wild type.
Dependence of mismatch binding activity on the concentration of p53
View larger version (56K):
[in a new window]
Fig. 3.
Mismatch binding activity of p53 determined
by gel retardation assays. A, gel retardation analysis
of p53 binding to lesion-free (WT), A/C, C/C, G/T,
3-cytosine bulge, and modGADD45 substrates was carried out by
incubating the DNA in the absence of p53 (lanes 1-7) or in
the presence of p53 (lanes 8-14) at a 1:10 ratio of DNA to
p53 (as tetramers) and a 20-fold excess of unlabeled nonspecific
competitor DNA. The positions of p53-DNA complexes (arrow
a), dsDNA (arrow b), and region of nonspecific binding
(bracket c) are indicated. B, histograms,
summarizing the results in A. Quantification of the amount
of DNA complexed with p53 was performed as described under
"Experimental Procedures."
View larger version (78K):
[in a new window]
Fig. 4.
Gel retardation analysis of p53 binding to
mismatched DNA using equal concentration of DNA. A, DNA
substrates (3 nM), 40 nM p53, and a 15-fold
excess of unlabeled dsDNA were incubated for 20 min at room temperature
prior to electrophoresis on 4% nondenaturing polyacrylamide gels.
Control reactions without p53 are not shown. Arrows a,
a', and a" denote specific p53-DNA complexes;
arrow b denotes dsDNA; and bracket c denotes the
area of nonspecific binding of p53 to DNA. B, histograms
summarizing the results from three separate gel analyses including the
assay shown in A. The y axis represents the fold
increase in p53 binding to mismatches compared with binding to wild
type (WT) DNA. Each bar represents the means ± S.E.
View larger version (21K):
[in a new window]
Fig. 5.
Affinity of p53 for different mismatched
substrates. Increasing amounts of mismatch containing DNAs were
incubated with a constant amount of p53 in the absence of nonspecific
competitor DNA. The protein-DNA complexes were separated on
nondenaturing polyacrylamide gels, and the amounts of free and bound
probe were quantified using a PhosphorImager. The curves
were drawn using Prism software as described in the text. A
shows the binding curves for substrates containing A/C, A/G, and C/T
mismatches. B shows the binding curves for substrates
containing G/T, 3-cytosine bulge, and A/A lesions. WT, wild
type.
View larger version (14K):
[in a new window]
Fig. 6.
Effect of increasing protein concentration on
p53 mismatch DNA binding activity. Gel retardation analysis was
performed for reactions containing 1 ng of DNA substrate and increasing
amounts of p53 in the absence of competitor DNA. DNA-p53 complexes were
quantified as described under "Experimental Procedures" and plotted
as a function of protein concentration. WT, wild type.
View larger version (17K):
[in a new window]
Fig. 7.
Competition analysis of p53 binding to
mismatched substrates. Labeled DNA substrates (1.5 nM
each) were incubated with 150 nM p53 for 20 min at room
temperature followed by addition of increasing amounts of unlabeled
nonspecific lesion-free (WT) competitor DNA for another 15 min prior to electrophoresis. The amount of DNA complexed with p53 was
plotted as a function of the amount of competitor DNA.
View larger version (66K):
[in a new window]
Fig. 8.
Comparison of the binding of p53 and
hMSH2-hMSH6 to mismatched substrates. A, DNA substrates
were incubated with p53 or hMSH2-hMSH6 at a 1:35 DNA to protein (p53
tetramers or hMSH2-hMSH6 heterodimers) molar ratio for 20 min at room
temperature in presence of a 15-fold excess of nonspecific competitor
DNA. The concentration of DNA used for each particular mismatched
substrate was identical for both proteins (0.3, 0.3, 1.2, 0.6, 0.3, 1.8, and 1.2 nM for lesion-free DNA, A/C, C/C, C/T, G/T,
3-cytosine bulge (BC), and GADD45-containing substrates,
respectively). Arrows indicate the products of the reaction:
arrows a, a', and a", p53-DNA
complexes; arrow b, hMSH2-hMSH6-DNA complexes; arrow
c, free dsDNA substrates. The hMSH2-hMSH6-DNA complexes have a
lower molecular mass than the p53-DNA complexes. B, specific
DNA-protein complexes were quantified as described under
"Experimental Procedures." The y axis represents the
percentage of DNA bound. WT, wild type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of the Griffith laboratory and Dr. Scott Gradia for helpful discussion, Dr. Arnold Levine and Dr. Richard Fishel for providing vectors for protein purification, and Drs. Michael Chernov and George Stark for two-dimensional gel analysis of our purified p53 protein.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA70343 and GM31819.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
919-966-2151; Fax: 919-966-3015; E-mail: jdg@med.unc.edu.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M006795200
2 M. Chernov and G. Stark, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ss, single-stranded; ds, double-stranded; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Agarwal, M. L.,
Taylor, W. R.,
Chernov, M. V.,
Chernova, O. B.,
and Stark, G. R.
(1998)
J. Biol. Chem.
273,
1-4 |
2. | Sionov, R. V., and Haupt, Y. (1999) Oncogene 18, 6145-6157[CrossRef][Medline] [Order article via Infotrieve] |
3. | Ko, L. J., and Prives, C. (1996) Genes Dev. 10, 1054-1072[CrossRef][Medline] [Order article via Infotrieve] |
4. | Levine, A. J. (1997) Cell 88, 323-331[Medline] [Order article via Infotrieve] |
5. | Oren, M., and Rotter, V. (1999) Cell. Mol. Life Sci. 55, 9-11[CrossRef][Medline] [Order article via Infotrieve] |
6. | Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W. (1991) Cancer Res. 51, 6304-6311[Abstract] |
7. | Nelson, W. G., and Kastan, M. B. (1994) Mol. Cell. Biol. 14, 1815-1823[Abstract] |
8. | Graeber, T. G., Peterson, J. F., Tsai, M., Monica, K., Fornace, A. J., Jr., and Giaccia, A. J. (1994) Mol. Cell. Biol. 14, 6264-6277[Abstract] |
9. | Linke, S. P., Clarkin, K. C., Di, Leonardo, A., Tsou, A., and Wahl, G. M. (1996) Genes Dev. 10, 934-947[Abstract] |
10. | Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe, S. W. (1997) Cell 88, 593-602[CrossRef][Medline] [Order article via Infotrieve] |
11. | el-Deiry, W. S. (1998) Semin. Cancer Biol. 8, 345-357[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Giaccia, A. J.,
and Kastan, M. B.
(1998)
Genes Dev.
12,
2973-2983 |
13. |
Sakaguchi, K.,
Herrera, J. E.,
Saito, S.,
Miki, T.,
Bustin, M.,
Vassilev, A.,
Anderson, C. W.,
and Appella, E.
(1998)
Genes Dev.
12,
2831-2841 |
14. | Jayaraman, L., and Prives, C. (1999) Cell. Mol. Life Sci. 55, 76-87[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hupp, T. R. (1999) Cell. Mol. Life Sci. 55, 88-95[CrossRef][Medline] [Order article via Infotrieve] |
16. |
McLure, K. G.,
and Lee, P. W.
(1998)
EMBO J.
17,
3342-3350 |
17. | Wang, X. W., and Harris, C. C. (1996) Cancer Surv. 28, 169-196[Medline] [Order article via Infotrieve] |
18. |
Spillare, E. A.,
Robles, A. I.,
Wang, X. W.,
Shen, J. C., Yu, C. E.,
Schellenberg, G. D.,
and Harris, C. C.
(1999)
Genes Dev.
13,
1355-1360 |
19. | Sturzbecher, H. W., Donzelmann, B., Henning, W., Knippschild, U., and Buchhop, S. (1996) EMBO J. 15, 1992-2002[Abstract] |
20. | Kuhn, C., Muller, F., Melle, C., Nasheuer, H. P., Janus, F., Deppert, W., and Grosse, F. (1999) Oncogene 18, 769-774[CrossRef][Medline] [Order article via Infotrieve] |
21. | Li, R., and Botchan, M. R. (1993) Cell 73, 1207-1221[Medline] [Order article via Infotrieve] |
22. | Wiesmuller, L., Cammenga, J., and Deppert, W. W. (1996) J. Virol. 70, 737-744[Abstract] |
23. |
Ford, J. M.,
and Hanawalt, P. C.
(1997)
J. Biol. Chem.
272,
28073-28080 |
24. | Bakalkin, G., Selivanova, G., Yakovleva, T., Kiseleva, E., Kashuba, E., Magnusson, K. P., Szekely, L., Klein, G., Terenius, L., and Wiman, K. G. (1995) Nucleic Acids Res. 23, 362-369[Abstract] |
25. |
Lee, S.,
Cavallo, L.,
and Griffith, J.
(1997)
J. Biol. Chem.
272,
7532-7539 |
26. |
Dudenhoffer, C.,
Rohaly, G.,
Will, K.,
Deppert, W.,
and Wiesmuller, L.
(1998)
Mol. Cell. Biol.
18,
5332-5342 |
27. | Lee, S., Elenbaas, B., Levine, A., and Griffith, J. (1995) Cell 81, 1013-1020[Medline] [Order article via Infotrieve] |
28. | Jayaraman, J., and Prives, C. (1995) Cell 81, 1021-1029[Medline] [Order article via Infotrieve] |
29. | Janus, F., Albrechtsen, N., Dornreiter, I., Wiesmuller, L., Grosse, F., and Deppert, W. (1999) Cell. Mol. Life Sci. 55, 12-27[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Okorokov, A. L.,
and Milner, J.
(1999)
Mol. Cell. Biol.
19,
7501-7510 |
31. |
Gradia, S.,
Acharya, S.,
and Fishel, R.
(2000)
J. Biol. Chem.
275,
3922-3930 |
32. | Fishel, R., Ewel, A., Lee, S., Lescoe, M. K., and Griffith, J. (1994) Science 266, 1403-1405[Medline] [Order article via Infotrieve] |
33. | Fishel, R., Ewel, A., and Lescoe, M. K. (1994) Cancer Res. 54, 5539-5542[Abstract] |
34. | Alani, E., Lee, S., Kane, M. F., Griffith, J., and Kolodner, R. D. (1997) J. Mol. Biol. 265, 289-301[CrossRef][Medline] [Order article via Infotrieve] |
35. | Rayssiguier, C., Thaler, D. S., and Radman, M. (1989) Nature 342, 396-401[CrossRef][Medline] [Order article via Infotrieve] |
36. | Worth, L., Jr., Clark, S., Radman, M., and Modrich, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3238-3241[Abstract] |
37. | Gamper, H., Lehman, N., Piette, J., and Hearst, J. E. (1985) DNA 4, 157-164[Medline] [Order article via Infotrieve] |
38. | Wu, L., Bayle, J. H., Elenbaas, B., Pavletich, N. P., and Levine, A. J. (1995) Mol. Cell. Biol. 15, 497-504[Abstract] |
39. | Gradia, S., Acharya, S., and Fishel, R. (1997) Cell 91, 995-1005[Medline] [Order article via Infotrieve] |
40. | Alani, E., Chi, N. W., and Kolodner, R. (1995) Genes Dev. 9, 234-247[Abstract] |
41. | Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606[Medline] [Order article via Infotrieve] |
42. | Bakalkin, G., Yakovleva, T., Selivanova, G., Magnusson, K. P., Szekely, L., Kiseleva, E., Klein, G., Terenius, L., and Wiman, K. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 413-417[Abstract] |
43. |
Jayaraman, L.,
Moorthy, N. C.,
Murthy, K. G.,
Manley, J. L.,
Bustin, M.,
and Prives, C.
(1998)
Genes Dev.
12,
462-472 |
44. | Mummenbrauer, T., Janus, F., Muller, B., Wiesmuller, L., Deppert, W., and Grosse, F. (1996) Cell 85, 1089-1099[Medline] [Order article via Infotrieve] |
45. | Steinmeyer, K., and Deppert, W. (1988) Oncogene 3, 501-507[Medline] [Order article via Infotrieve] |
46. |
Acharya, S.,
Wilson, T.,
Gradia, S.,
Kane, M. F.,
Guerrette, S.,
Marsischky, G. T.,
Kolodner, R.,
and Fishel, R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13629-13634 |
47. | Marsischky, G. T., Filosi, N., Kane, M. F., and Kolodner, R. (1996) Genes Dev. 10, 407-420[Abstract] |
48. |
Wang, Y. H.,
Bortner, C. D.,
and Griffith, J.
(1993)
J. Biol. Chem.
268,
17571-17577 |