Download Maintenance of genomic integrity by p53: complementary

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Oncogene (1999) 18, 7706 ± 7717
1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00
http://www.stockton-press.co.uk/onc
Maintenance of genomic integrity by p53: complementary roles for
activated and non-activated p53
Nils Albrechtsen1, Irene Dornreiter1, Frank Grosse2, Ella Kim1, Lisa WiesmuÈller1 and
Wolfgang Deppert*,1
1
Heinrich-Pette-Institut fuÈr Experimentelle Virologie und Immunologie an der UniversitaÈt Hamburg, Martinistrasse 52, D-20251
Hamburg, Germany; 2Institut fuÈr Molekulare Biotechnologie, *Abt. Biochemie, Beutenbergstr. 11, D-07745 Jena, Germany
In this review we describe the multiple functions of p53 in
response to DNA damage, with an emphasis on p53's
role in DNA repair. We summarize data demonstrating
that p53, through its various biochemical activities and
via its ability to interact with components of the repair
and recombination machinery, actively participates in
various processes of DNA repair and DNA recombination. An important aspect in evaluating p53 functions
arises from the ®nding that the p53 core domain harbors
two mutually exclusive biochemical activities, sequencespeci®c DNA binding, required for its transactivation
function, and 3'-45' exonuclease activity, possibly
involved in various aspects of DNA repair. As modi®cations of p53 that lead to activation of its sequencespeci®c DNA-binding activity result in inactivation of its
3'-4 5' exonuclease activity, we propose that p53 exerts
its functions as a `guardian of the genome' at various
levels: in its non-induced state, p53 should not be
regarded as a non-functional protein, but might be
actively involved in prevention and repair of endogenous
DNA damage, for example via its exonuclease activity.
Upon induction through exogenous DNA damage, p53
will exert its well-documented functions as a superior
response element in various types of cellular stress. The
dual role model for p53 in maintaining genomic integrity
signi®cantly enhances p53's possibilities as a guardian of
the genome.
Keywords: p53; DNA repair; DNA damage; DNA
recombination; DNA replication; sequence-speci®c
DNA binding
Introduction
Twenty years after its discovery, the tumor suppressor
p53 has become one of the most prominent molecules
in the area of cancer research. Initially classi®ed as an
oncogene (reviewed in Deppert, 1994) p53 meanwhile is
regarded as a `master tumor suppressor' which ensures
the integrity of the cells' genome by protecting it from
adverse e€ects of DNA damage. Such damage triggers
the activation and accumulation of p53 (Fritsche et al.,
1993; Kastan et al., 1991; Nelson and Kastan, 1994).
The best studied function of an activated p53 is that of
a transcription factor, mediating expression of a variety
of genes that induce a growth arrest or apoptosis. As a
*Correspondence: W Deppert
result, DNA damage will be repaired during a growth
arrest, or the damaged cells will be eliminated, thereby
preventing ®xation of DNA damage as mutations. This
function of p53 led to the now famous coining of p53
as the `guardian of the genome' by Lane (1992).
Although the main features of p53's role in
maintaining the integrity of the genome seem to be
outlined, there is still a lot to be learned as to how p53
exactly acts to prevent the accumulation of mutational
events within a cell. Despite some uncertainties
regarding the various mechanisms of its induction,
elimination of damaged cells by p53-induced apoptosis
is an easily conceived mechanism for achieving this
goal. Much less is known about the role of p53 in
DNA repair. So far, the main emphasis has been given
to pathways leading to the activation of p53 by signals
emanating from damaged DNA, with p53 integrating
these signals and triggering a cascade of responses
leading to either growth arrest or apoptosis. These
mechanisms have been summarized in detail in several
recent reviews (for example see Cox and Lane, 1995;
Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine,
1997; Bates and Vousden, 1999), thus this topic will be
addressed here only brie¯y. The next steps in this
cascade, namely the activation of the repair pathways
themselves, and the role p53 plays in their activation
are far less clear. Furthermore, it is still not known,
whether and how p53 directly participates in DNA
repair processes, despite some evidence pointing to this
possibility. Last but not least, a possible role of p53 in
the control of genomic integrity in its non-induced
state, i.e., in the absence of signals indicating DNA
damage or other potentially harmful conditions, so far
has not yet been considered at all. In contrast, the
general assumption is that a non-activated p53 is a
non-functional one (Hupp and Lane, 1994; Vogelstein
and Kinzler, 1992).
In this review we will focus on p53-induced
pathways that lead to or are part of DNA repair
processes, with an emphasis on the known biochemical
activities of p53, which might play a direct role in
repair processes. Recent evidence from our laboratory
showed that at least one of these activities, the 3'-45'
exonuclease activity, is regulated in a manner opposite
to that of sequence-speci®c DNA binding (Janus et al.,
1999). Based on this ®nding we propose a model
according to which p53 in its non-induced state, i.e. in
the absence of conditions which lead to its activation,
exerts basic functions in maintaining genomic integrity.
Upon various cellular stress conditions, such as
hypoxia, nutrient depletion, or DNA damage, the
well known functions of p53 in integrating the
respective signals become activated, triggering the
Dual role model for p53 function
N Albrechtsen et al
cascade of p53 responses which lead to growth arrest,
DNA repair, or apoptosis (Hall et al., 1996).
Activation of p53 upon DNA damage
Cell cycle check point control and apoptosis
Eukaryotic cells have developed a network of highly
conserved surveillance mechanisms (checkpoints) which
ensure that damaged chromosomes are repaired before
they are replicated or segregated. These mechanisms
are essential for maintaining genomic integrity and cell
viability. The tumor suppressor p53 is one of the
critical mediators of cellular responses to various types
of genotoxic stress (reviewed e.g. in Hall et al., 1996),
acting at di€erent levels of control during the cell cycle.
In response to DNA damage, like ionizing irradiation (IR) (Kastan et al., 1991), ultraviolet (UV)
irradiation (Maltzman and Czyzyk, 1984; Nelson and
Kastan, 1994), hypoxia (Graeber et al., 1994), and
ribonucleoside triphosphate depletion (Linke et al.,
1996), p53 becomes activated as a transcription factor
via speci®c post-translational modi®cations. The p53
protein is phosphorylated by a wide variety of protein
kinases: in its transactivation domain by casein kinase
(Ck) I, DNA-PK, ATM, JNK, and MAP kinases, and
at its C terminus by cyclin dependent kinases (Cdks),
PKC, and Ck II (see Martinez et al., 1997; Meek, 1994
and references herein). The di€erent protein kinases
can modify the sequence-speci®c DNA binding activity
of p53 and may thereby modulate the relative eciency
of activation of di€erent p53 target genes (Hecker et
al., 1996; Hupp and Lane, 1994; Hupp et al., 1992;
Takenaka et al., 1995; Wang and Prives, 1995).
Phosphorylation by DNA-PK within the transactivation domain of the protein (Shieh et al., 1997) also
abrogates its interaction with MDM2, a key player in
negative regulation of the transcriptional activity and
the level of p53 (reviewed in Lane and Hall, 1997).
DNA-PK phosphorylation on serine 15 and possibly
serine 37 within (human) p53 correlates with the
enhanced transcription of downstream p53 target
genes. The ability of p53 to transactivate downstream
target genes results in cell cycle arrest at speci®c points
in the cell cycle. As a consequence, the cell cycle stops
either before DNA replication in G1, or before mitosis
in G2, which leads to a severe decrease in the amount
of S phase cells.
G1 arrest The p53-dependent G1 arrest results mainly
from transactivation of the waf1 gene that codes for
the small kinase inhibitor p21Waf1. p21Waf1 interferes with
cell-cycle progression and prevents S phase entry by
blocking the activity of Cdks (Dulic et al., 1994; ElDeiry et al., 1993; Harper et al., 1993; Xiong et al.,
1993). Irradiated G1 phase cells accumulate high levels
of Cdk2/cyclin E complexes which are inactivated by
the association with p21Waf1. Inhibition of G1 phasespeci®c kinase activity maintains a hypophosphorylated
retinoblastoma susceptibility gene product pRb, which
represses E2F-speci®c transcription of genes that are
required for entry into S phase, and thereby inhibit cell
cycle progression.
p53 may also be able to induce a G1-arrest by nontranscriptional mechanisms. Recently, it has been
observed that p53 binds cyclin H, which is part of
the Cdk7/cyclinH/Mat1 Cdk activating kinase complex
(CAK). CAK plays a crucial role in activating cell
cycle progression by phosphorylation and activation of
Cdk2, and, as part of the TFHII complex, by
controlling transcriptional activity of RNA polymerase II. In the TFHII complex, CAK phosphorylates the
carboxy-terminal repeat domain (CTD) of RNA
polymerase II, which is required for the transcriptional
elongation by RNA polymerase II. Binding of p53 to
CAK results in strong reduction of CAK activity both
towards cdk2 and towards the CTD of RNA
polymerase II, which could lead to either growth
arrest or apoptosis (Ko et al., 1997; Schneider et al.,
1998).
S phase arrest In another checkpoint response pathway which operates in S phase and slows down the rate
of DNA replication, p21Waf1 binds to the proliferatingcell nuclear antigen (PCNA) and blocks its activity,
interfering with cell-cycle progression by blocking the
elongation step in DNA replication (Waga et al., 1994).
Stillman and coworkers suggested a dual role for
PCNA in DNA replication and DNA repair which
allows p21Waf1 to arrest DNA replication while
permitting active DNA repair (Li et al., 1994a). In
DNA replication, PCNA together with replication
factor C (RFC) recognizes a primer-template junction
and promotes loading of polymerase d (pol d). The
PCNA-RFC-pol d complex also enhances the processivity of pol d during the elongation step of DNA
replication (Melendy and Stillman, 1991). The direct
binding of p21Waf1 to PCNA causes a rapid dissociation
of the PCNA-RFC-pol d complex from the replication
fork, stalling replicative DNA synthesis. In repair
DNA synthesis, PCNA helps the polymerases d or e
to localize the junction of incised DNA, but is not
needed during DNA synthesis (Li et al., 1994a).
Thereby, repair DNA synthesis could proceed also in
the presence of an upregulated p21Waf1.
G2 arrest The third and the last line of defense
against induced chromatid damage occurs in G2. A
possible role for p53 in the G2/M checkpoint was
suggested by Li et al. (1994b) who reported that p21Waf1
can associate with cyclin A and cyclin B complexes
during the later phases of the cell cycle, suggesting a
functional interaction with the respective associated
kinases. Furthermore, a bimodal periodicity for waf1
mRNA levels in human ®broblasts with peaks in G1
and G2/M was observed, proposing that p21Waf1 may
play a role at the onset of mitosis. p21Waf1 is absent
from the nucleus during S phase and transiently
reenters the nucleus during late G2 phase. In late G2
half of the Cdk2/cyclin A is complexed with p21Waf1.
p21Waf1 may thereby either directly inhibit the active
kinase or prevent its activation by the Cdk-activating
kinase CAK (Poon et al., 1996). Another possibility is
that complex formation with p21Waf1 may block
interaction of substrates with Cdk2/cylin A (Adams
et al., 1996). In Xenopus egg extracts Cdk2 serves as a
positive regulator for the activation of Cdk1/cyclin B
complexes and prevents entry into mitosis when
complexed to p21Waf1 (Guadagno and Newport, 1996).
Nuclear accumulation of p21Waf1 and the resulting
inactivation of Cdk-cyclin complexes at the onset of
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N Albrechtsen et al
7708
mitosis might be part of a p21Waf1 dependent mitotic
attenuation mechanism (Dulic et al., 1998). However,
p21Waf1 accumulation resulting from p53 activation
seems to be mainly required for the DNA damageinduced G1 arrest, as p21Waf1 clearly is not essential for
the immediate G2 checkpoint response (Brugarolas et
al., 1995; Levedakou et al., 1995; Paules et al., 1995).
Nevertheless, several studies have suggested that the
G2/M arrest following DNA damage is also p53dependent (Agarwal et al., 1995; Aloni-Grinstein et al.,
1995; Goi et al., 1997; Schwartz et al., 1997; Stewart et
al., 1995).
Vogelstein and colleagues used a wild-type p53
expressing human colorectal cancer cell line to analyse
gene expression following g-irradiation. The cells
arrested mostly in G2, and the block was accompanied
by changes in gene expression. Quantitative analysis of
gene expression patterns revealed a strong induction of
the 14.3.3s gene. The induction of 14.3.3s is mediated
by a p53-responsive element in the 14.3.3s gene
(Hermeking et al., 1997). The 14.3.3-family proteins
are found in a wide variety of mammalian tissues and
in other eukaryotic organisms including plants and
yeast (Aitken et al., 1992), and diverse biochemical
properties have been ascribed to them. Mammalian
cells contain a minimum of seven 14.3.3 isoforms
(Aitken et al., 1995; Wang and Shakes, 1996). Studies
in yeast indicate that 14.3.3 proteins have a role in
determining the timing of mitosis (Ford et al., 1994).
The induction of 14.3.3s by p53 might be part of p53's
control of G2/M transition. The link between DNA
damage control and cell cycle checkpoint control at
G2/M could be further substantiated by the following
observations. In irradiated cells the protein kinase
Chk1, which is required for this DNA damage
checkpoint (Sanchez et al., 1997), undergoes a Rad3dependent phosphorylation (Rad3 is a kinase related to
the ATM protein) (Ford et al., 1994). The ultimate
target of this checkpoint signal is thought to be Cdk1,
the cyclin-dependent kinase that induces mitosis (Pines,
1991), as Chk1 directly phopshorylates Cdc25C, a
phosphatase that regulates Cdk1 activity (Furnari et
al., 1997; Peng et al., 1997; Sanchez et al., 1997). This
phosphorylation event promotes the binding of 14.3.3
to Cdc25C and thereby its sequestration. In this state
Cdc25C cannot dephosphorylate and activate Cdk1.
Thus cell cycle progression after g-irradiation (and
possibly other DNA damage inducing events) is
blocked by independent p53-mediated e€ector pathways: Entry into S phase is inhibited by p21Waf1,
whereas 14.3.3s prevents cells that have completed S
phase from entering mitosis. Therefore the p53dependent induction of 14.3.3s connects DNA
damage with G2/M progression driven by Cdk1 in
the same way as the p53-dependent induction of p21Waf1
connects DNA damage with the Cdks required for G1/
S progression.
Apoptosis In response to DNA damage, p53 can also
induce apoptosis. Induction of apoptosis by p53 is
complex and not yet understood in detail. Transcriptional activation of a variety of di€erent genes involved
in mediating an apoptotic response (e.g. bax, Fas/Apo,
Killer/DR5, and redox regulator genes [PIGs]) again
seems to be the major mechanism of p53-mediated
apoptosis, but p53 is able to induce apoptosis also in a
transcription-independent mode (Caelles et al., 1994;
Haupt et al., 1995; reviewed in Bates and Vousden,
1999). The interaction of p53 with CAK already
described above could be one such mechanism, but
other interactions, mediated by the N-terminal prolinerich domain of p53 which serves as a docking place for
proteins containing SH3-domains, have also been
implicated.
Even less understood is what determines the
`decision' to induce either a growth arrest or apoptosis
after p53 activation. The outcome de®nitely is cell type
speci®c, and is also regulated by the severity of DNA
damage, with heavier damage promoting apoptosis
over growth arrest. Induction of growth arrest rather
than apoptosis on the other hand is favored by the
presence of survival factors. In this respect, it is
interesting that IGF-BP3 is a target for transcriptional
activation by p53. Overexpression of IGF-BP3 can
inhibit the mitogenic activity of IGF receptor signaling,
while in other systems it can inhibit the survival signals
provided by IGF, thereby amplifying apoptosis. Thus
the extracellular environment and intracellular signals
strongly modulate the p53 response (reviewed in Bates
and Vousden, 1999).
Selectivity of transcriptional regulation by p53 As the
outcome of the cellular response upon activation of
p53 to a great extent is dependent on transactivation of
the appropriate target genes, the question arises how
selectivity of transactivation is secured. So far,
approximately 20 more or less con®rmed target genes
of p53 have been described, which will induce quite
di€erently and even opposing or mutually exclusive
cellular responses. Thus it is necessary that p53 is able
to discriminate between those genes, activating
transcription from only those target genes which
mediate the appropriate response in a given cellular
environment and physiological situation. Several ways
to achieve this goal can be envisioned. It has been
proposed that di€erent posttranslational modi®cations
of the p53 protein will lead to the preferential
activation of certain target genes (reviewed in Jayaraman and Prives, 1999). Similarly, the association of p53
with di€erent co-activator molecules will confer
speci®city and selectivity to p53-mediated transactivation. For example, association of p33ING with p53
seems to be required for the ecient transactivation of
the waf1-promoter by p53 (Garkavtsev et al., 1998). In
addition to these mechanisms our laboratory recently
suggested a novel way to ensure speci®city and
selectivity of transactivation by p53. The cognate
DNA sequences in p53-responsive promoters are
characterized by signi®cant sequence-heterogeneity, as
the consensus sequence, consisting of two 5'-PuPuPuC(A/T)(TA)GPyPyPy-3' half-sites separated by 0 ±
13 bp (EI-Deiry et al., 1992), by itself allows for many
variations. In addition, analysis of promoter sequences
transactivated by p53 showed that even the presence of
several bases not conforming to the consensus is
tolerated (Bargonetti et al., 1993; Foord et al., 1993).
This amazing heterogeneity within p53 cognate sites
asks for additional mechanisms providing speci®city
and selectivity to sequence-speci®c transactivation by
p53. Indeed, close examination of the various natural
and arti®cial DNA sequences recognized by p53
demonstrated that, despite their sequence-heterogene-
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N Albrechtsen et al
ity, many of them display an internal symmetry as a
common feature. Internal symmetry is a hallmark of
DNA sequences able to assume a non-B-DNA
conformation upon topological stress. We therefore
speculated whether p53 promoter DNA might assume
di€erent conformational states, and whether p53 might
be able to interact with such DNA. Using a variety of
arti®cial DNA substrates displaying the same p53
target sequence in di€erent conformational states, we
were able to demonstrate that p53 indeed is able to
recognize isoforms of the same target sequence
presented in di€erent conformations. Interestingly,
recognition of such isoforms strongly depended on
p53 protein conformation: whereas recognition of
isoforms in duplex B-DNA conformation required
activation of p53 by addition of PAb421, recognition
of isoforms in non-B-DNA conformations did not. On
the contrary, binding of p53 to some of such isoforms
was inhibited by PAb421. We concluded that sequencespeci®c DNA binding of p53 can be modulated both
by p53 protein conformation and by the conformation
of its cognate DNA sequence (Kim et al., 1997).
This ®nding opens the way for a better understanding of target selectivity of p53 sequence-speci®c
transactivation, as it is becoming increasingly accepted
that chromatin con®guration, speci®cally promoter
DNA conformation, is an important determinant of
promoter activation. Accordingly, the promoter DNA
can be present in an active conformation, which allows
transcription, and an inactive conformation, which
prohibits transcription. We suggest that p53-reactive
promoters can adopt such conformations, and that
promoter DNA topology and p53 protein conformation (and/or interaction with auxiliary proteins)
determine the interaction of p53 with target gene
promoters. In support of this model, we were able to
show that e.g. recognition of the mdm2-promoter by
p53 is impaired, when this promoter assumes a non-B
DNA conformation in superhelical DNA. Relaxation
of superhelicity by topoisomerase treatment restored
p53 binding and increased the level of sequence-speci®c
transactivation by p53 (Kim et al., submitted). In vivo,
conformational changes in chromatin are mediated by
proteins modeling chromatin architecture (Nickerson,
1998; Zlatanova and van Holde, 1998). With regard to
our model it therefore may be more than coincidence
that p53 interacts with at least two proteins involved in
such processes, HMG1 (Jayaraman et al., 1998), and
topoisomerase I (Gobert et al., 1996). Another protein
that is involved in chromatin remodeling and p53
binding is the transcriptional co-activator CBP/p300.
This protein forms a complex with p53, which in turn
is able to enhance transcription of p53-dependent genes
(Avantaggiati et al., 1997; Gu and Roeder, 1997). The
interaction of CBP and p53 is strongly stimulated after
phosphorylation of p53 at serine 15 (Lambert et al.,
1998). CBP/p300 bears an intrinsic histone acetyltransferase (HAT) activity that is thought to be involved in
histone acetylation and chromatin decondensation
(Bannister and Kouzarides, 1996; Ogryzko et al.,
1996). The HAT activity of CBP/p300 is controlled
by cell cycle-dependent kinases and the oncoprotein
E1A (Ait-Si-Ali et al., 1998). The HAT activity also
phosphorylates p53 in vitro and thereby increases its
transcriptional potential (Sakaguchi et al., 1998). HATmediated acetylation of p53 seems also to occur in vivo,
particularly in response to DNA damage (Liu et al.,
1999). Hence it appears that there exists another
regulatory circuit that consists of HATs, p53, and the
reorganization of the chromatin structure, that may
respond to physiological situations such as the ongoing
cell cycle and also to pathological threats, such as
DNA damage.
DNA repair after activation of p53 by DNA damage
As outlined above, activation of p53 after DNA
damage leads to a p53-induced growth arrest or
apoptosis. Whereas induction of apoptosis bypasses
any need to repair DNA damage, a p53-induced
transient growth arrest will ful®ll its intention only, if
it achieves an accurate repair of the damaged DNA.
As sequence-speci®c transactivation of p53 target
genes is regarded as the major function of p53 after
its activation, upregulation of genes involved in DNA
repair might be envisioned as a ®rst step to initiate
repair of DNA damage. However, with the exception
of the gadd45 gene (Kastan et al., 1992), and the p48
xeroderma pigmentosum gene (Hwang et al., 1999),
which have been linked to DNA repair, no such
direct e€ect of p53 is known. One thus has to
conclude that DNA repair during a p53-induced
growth arrest occurs in a p53-independent manner, or
alternatively, that p53 participates in DNA repair by
other activities. Although there is de®nitely p53independent DNA repair (as can be demonstrated
in p53-de®cient cells), there is also evidence for a
participation of p53 in repair processes, and for a
p53-dependent activation of DNA repair. The
assumption of a direct involvement of p53 in DNA
repair leads to the prediction that mutations in p53
or the lack of p53 should lead to a defect in DNA
repair. If p53 actively participates in DNA repair,
p53 should interact with proteins that are part of
DNA repair pathways and/or possess biochemical
activities by which it could be part of repair
pathways.
To test for a direct involvement of p53 in DNA
repair, Ford and Hanawalt examined Li-Fraumeni
®broblasts which were homozygous for a mutated
p53 and found that these cells were indeed de®cient in
the rate and extent of nuclear excision repair (NER) of
genomic DNA (Ford and Hanawalt, 1995, 1997). In
contrast, they did not observe a defect in transcriptioncoupled NER. This point, however, remains somewhat
controversial, since other investigators found a
de®ciency in transcription-coupled NER (Mirzayans
et al., 1996; Wang et al., 1995). Reduced NER was also
observed when the normal p53 function was disrupted
by targeting p53 for degradation by the human
papillomavirus E6 oncogene, or by expression of a
dominant-negative mutant p53 (Smith et al., 1995).
Furthermore, Li-Fraumeni-Syndrome cells with mutated p53 are impaired in their recovery for both RNA
and DNA synthesis after UV-treatment, an anomaly
which they share with the DNA repair disorders
xeroderma pigmentosum and Cockayne's syndrome
(Mirzayans et al., 1996). In this respect, p53 behaves
like other proteins that are directly involved in DNA
repair. But not only does a defect in p53 function result
in a defective DNA repair, the induction of p53 after a
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N Albrechtsen et al
7710
treatment mimicking UV-damage even leads to an
enhancement of DNA repair (Eller et al., 1997).
Association of p53 with components of repair pathways
Data showing a speci®c interaction of p53 with
components of repair pathways provided clues as to
how p53 is involved in DNA repair. Most of these
proteins are members of the transcription factor IIH
(TFIIH) multiprotein complex, which initiates basal
transcription of RNA polymerase II and couples
transcription with NER) (Leveillard et al., 1996;
Wang et al., 1995; Xiao et al., 1994). p53 interacts
with
three
components
(XPB=ERCC3,
XPD=ERCC2 and p62) of the TFIIH protein
complex. p53 inhibits the helicase activity of XPB
and XPD, probably through its strand-reannealing
properties (Leveillard et al., 1996; Wang et al., 1995).
The e€ect of TFIIH on p53 function is not yet known.
It is possible that the interaction of TFIIH components
with p53 serves to colocalize p53 at sites of DNA
repair or transcriptional initiation.
p53 also binds to Cockayne syndrome B protein
(CSB), which was identi®ed as a repair protein by
phenotypic complementation of an excision repairde®cient rodent cell line (Troelstra et al., 1990). The
encoded protein is functionally defect in human
Cockayne syndrome type B, the most common form
of this disease. A‚icted individuals are mentally
retarded and sensitive to sunlight. Wang and
colleagues were able to show that in vitro translated
CSB also binds speci®cally to GST-wild type p53
fusion protein (Wang et al., 1995). CSB, like ERCC2
and ERCC3 also possesses an unwinding activity.
Therefore, binding of p53 to helicases involved in NER
(and possibly their inhibition) seems to be a common
feature of p53, although no mechanistic explanation
has been provided so far. Another p53-binding partner
involved in DNA repair is the single-stranded DNA
binding protein RPA. UV irradiation disrupts the
RPA-p53 complex, correlating with an activation of
p53, but only in cells which are capable to carry out
global nucleotide excision repair (Abramova et al.,
1997). Since UV-irradiation is a major signal for the
activation and induction of p53, it seems likely that
binding of p53 to RPA is involved in upstream
regulation of p53-dependent damage response. Combined with the fact that a defect in p53 function leads
to a defect in global NER, it may be more than
coincidence that UV-irradiation leads to a disruption
of the binding of RPA to p53 only in cells capable of
global NER. A plausible model is that RPA in nonirradiated cells sequesters p53, but releases it after
DNA damage. The released p53 could then act as a
transcriptional activator, or/and could directly participate in global NER.
Biochemical activities of p53 related to DNA repair
An important feature of p53 in considering its direct
involvement in DNA repair is its ability to interact
with DNA in many di€erent ways: p53 binds to
double-stranded and single-stranded DNA in a nonsequence-speci®c manner (Steinmeyer and Deppert,
1988), to ends of double-strand breaks (Bakalkin et
al., 1995), to Holliday junctions (Lee et al., 1997), and
to DNA mismatches leading to DNA-`bulges' (Lee et
al., 1995). These activities are important for p53's
ability to bind to damaged DNA. p53 thus can `sense'
and bind strongly to DNA damaged by ionizing
radiation (Reed et al., 1995) and form highly stable
complexes with insertion/deletion mismatches (Lee et
al., 1995). p53 in this respect shows parallels to the
DNA repair factor MSH2. Binding to damaged DNA
is mediated by the C-terminal domain of p53, which
has been shown to be important for the regulation of
p53 function. The `sensing' mechanism forms the basis
for a model in which p53 via its C-terminus recognizes
DNA damage. p53 then becomes activated for
sequence-speci®c DNA binding and for transactivation of target genes, which then participate in and
enhance DNA repair (Jayaraman and Prives, 1995).
However, this model is confronted with a major
problem: it is dicult to envision how p53 will bind
tightly to damaged DNA with a half-life of more than
2 h (Lee et al., 1995), and at the same time will
transactivate genes which in all likelihood are located
in a distant part of the genome, i.e. megabases away
from the DNA damage locus. In addition, as already
indicated above, so far no p53 target genes have been
identi®ed, which could account for the global repair
defect when p53 is mutated. Therefore we suggest that
p53, in addition to damage recognition, could be
directly involved in DNA repair processes by several
biochemical activities, namely by its non-sequencespeci®c DNA binding activity, its DNA reannealing
activity, its ability to promote DNA strand transfer,
and its 3'-45' exonuclease activity. Especially the p53
intrinsic 3'-45' exonuclease activity, localized within
the p53 core domain (Mummenbrauer et al., 1996);
could be an important player in repair activities of p53.
Exonucleases are required for DNA replication, DNA
repair and recombination and often enhance the
®delity of these processes. As mutant p53 is
exonuclease de®cient (Mummenbrauer et al., 1996),
and cells expressing a mutant p53 are defective in
global NER (Ford and Hanawalt, 1995, 1997; Wang
and Prives, 1995; Mirzayans et al., 1996), this
correlation might point to a possible role of the p53
exonuclease activity in DNA repair. The various
biochemical activities of p53 and its interactions with
viral and cellular proteins are summarized and related
to p53 structure in Figure 1.
Involvement of p53 in control of homologous
recombination
Recombination processes are subject to complex
surveillance mechanisms ensuring high ®delity of
DNA repair and of genetic transmission during
meiosis (Haber, 1997; Xu et al., 1997). Regulatory
circuits must also exist in order to establish di€erences
in DNA exchange frequencies between distinct genomic
loci during meiosis, and in mitotically growing cells,
where homologous recombination must be suppressed
by a factor of 1000. p53 accumulates and becomes
functionally activated (Lutzker and Levine, 1996;
Siegel et al., 1995) upon the introduction of double
strand breaks (DSB) into DNA (Nelson and Kastan,
1994). DSB arise spontaneously due to errors in
replication, recombination or mitosis and can be
Dual role model for p53 function
N Albrechtsen et al
7711
Figure 1 p53 landmarks. Roman numerals represent the ®ve regions of p53 that are conserved within p53 from all vertebrates.
Known phosphorylation (P) and acetylation (Ac) sites are indicated. The vertical bars, clustered in the center of the p53 molecule,
indicate amino acid residues mutated in human tumors (hot spots are identi®ed by amino acid number). Shown below and indicated
by horizontal bars is the current information concerning various domains of p53 for biological activities, p53 DNA interactions, and
p53-protein complex formation. Abbreviations: CK 2, casein kinase 2; CSB, Cockayne's syndrome B protein; DNA PK, DNA
dependent protein kinase; NLS, main nuclear localization signal; RP-A, replication protein A; SV 40, Simian Virus 40; TAF,
transcription activating factor; TBP, TATA-Box binding protein; TF, transcription factor; XPB, xeroderma pigmentosum B protein;
XPD, xeroderma pigmentosum D protein
induced experimentally by ionizing radiation, radiomimetic drugs, or by the introduction of restriction
endonucleases. DSB trigger both repair associated and
targeted recombination processes, a connection which
in turn renders p53 a likely candidate for being a
regulatory factor in DNA exchange processes. During
the last years we (WiesmuÈller et al., 1996) and others
(Mekeel et al., 1997; Bertrand et al., 1997; Honma et
al., 1997) demonstrated that p53 suppresses spontaneous inter- and intrachromosomal homologous
recombination events at least by one to two orders of
magnitude. The same groups also demonstrated that
transcriptional transactivation and cell cycle control
activities are separable from p53's involvement in
homologous recombination by comparing the activities of cancer-related p53 mutants and C-terminally
truncated p53 variants in these processes (DudenhoȀer
et al., 1999; BS Lopez, Paris, and SN Powell, personal
communication).
Inhibition of p53 recombination control by SV40
SV40 provides a suitable model system for probing
recombination, taking advantage of the small
chromatin-packaged viral genome, which is ampli®ed episomally, and which promotes high frequency
exchange rates for easy detection of recombination
events (WiesmuÈller et al., 1996; Jasin et al., 1985;
Snapka et al., 1991; Kawasaki et al., 1994). The
SV40 tumor antigen (T-Ag) was found to be the
causative agent for the elevated recombination
frequencies of cellular and viral DNAs as well as
for the stimulation of the closely related gene
ampli®cation events (WiesmuÈller et al., 1996; Perry
et al., 1992; Ishizaka et al., 1995; Cheng et al.,
1997). SV40 T-Ag targets p53 in SV40 infection and
transformation by forming a tight complex with p53.
A possible role for p53-T-Ag complex formation is
the elimination of a function of p53 in the control
of recombination. By comparing the recombination
rates between SV40 genomes expressing a wild type
T-Ag or a T-Ag containing a point mutation which
speci®cally abolishes T-Ag-p53 interactions (WiesmuÈller et al., 1996), we found that recombination
frequencies in cells expressing a T-Ag unable to bind
p53 were reduced by at least one order of
magnitude. Therefore we conclude that suppression
of homologous recombination events by wild type
p53 can be alleviated by complex formation with
SV40 T-Ag (WiesmuÈller et al., 1996).
Elimination of p53-mediated suppression of recombination by T-Ag might also explain the ®nding that
immortalization of human ®broblasts after stable
transformation with T-Ag increases chromosomal
recombination. Other viral binding partners, such as
HPV16 E6 (Mekeel et al., 1997; Havre et al., 1995),
Dual role model for p53 function
N Albrechtsen et al
7712
and possibly the genetically destabilizing Hepatitis B
virus (HBV) X antigen (Feitelson et al., 1993), may
also neutralize the inhibitory function of p53 in
recombination, thereby allowing a correlation between
the targeting of p53 by these proteins and elevated
recombination.
Mismatch-recognition: A speci®c role for p53 in
recombination control?
We recently discovered that p53 inhibits DNA
exchange most dramatically upon recognition of
certain mismatches within nascent recombination
intermediates (Dudenhoe€er et al., 1998). Parallels
to the functions of classical mismatch repair factors
in recombination arise, as the mammalian MutS
counterpart, MSH2, like p53, binds to Holliday
junctions (Alani et al., 1997) and abolishes DNA
exchange between divergent sequences (De Wind et
al., 1995). In support of our idea, p53 was shown to
equally a€ect homologous recombination between
direct and indirect repeat substrates, which allows
the conclusion that p53 controls recombination at
the stage of strand invasion (BS Lopez, personal
communication). Therefore, it is tempting to
speculate that p53 monitors the ®delity of recombination in concert with the mismatch repair system.
MSH2 and p53 do not seem to perform structural
but rather regulatory functions during DNAexchange processes. Consistent with the hypothesis
of two surveillance pathways acting in parallel, the
inactivation of both pathways causes a synergism in
tumorigenesis, evidence for which was recently given
by the analysis of Msh2 and p53 double knock-out
mice (Cranston et al., 1997). Therefore, MSH2 and
p53 must be considered to be members of di€erent
genetic epistasis groups involved in the regulation of
recombination processes. The view is consistent with
our biochemical data suggesting complementary
®delity control due to an opposing mismatch
speci®city for MSH2-GT-binding protein complexes,
which most eciently recognize G-T single base
mispairings (Hughes and Jiricny, 1992), and for p53tetramers, which display highest anities for A ± G
mismatches (Dudenhoe€er et al., 1998). It remains
to be established, whether p53 upon encountering
mismatches in heteroduplices transmits signals to
downstream molecules, such as p21waf1, or/and blocks
the progress of DNA exchange, possibly even by
participating in error removal through its 3'-45'
exonuclease activity. First indications for a p53mediated signal transduction mechanism coupled to
recombination processes can be derived from the
fact that the hRad51-complex partners Brca1 and
Brca2 physically and genetically interact with p53
(StuÈrzbecher et al., 1996; Ouchi et al., 1998; Zhang
et al., 1998; Lim and Hasty, 1996; Hakem et al.,
1997; Ludwig et al., 1997), and that Brca1 seems to
stimulate p53-dependent transcription, whereas overexpressed Brca2 and hRad51 display antagonistic
e€ects (Marmorstein et al., 1998). In line with this
proposal is the observation that puri®ed multiprotein
complexes performing homologous recombination in
vitro contain DNA polymerase e, DNA ligase and
low levels of 5'-43' and 3'-45' exonuclease activities
(Jessberger et al., 1993).
Is there a role for p53 in DNA repair in its non-induced
state?
The transactivation and the exonuclease function of p53
seem to be mutually exclusive
The active participation of p53 in DNA repair
processes might not be restricted to situations
activating a p53 response, like DNA damage in¯icted
by exogenous factors. On the contrary, expanding
p53's role as `guardian of the genome' by endowing it
with a basic function in DNA repair processes in its
non-induced state instead of being just a nonfunctional protein should greatly enhance its possibilities to preserve the integrity of the genome. Actually,
some inconsistencies regarding the role of the waf1
gene as a major target in the p53 response to DNA
damage already point to such a basic function. If
indeed p53-mediated induction of the waf1 gene, and
the ensuing growth arrest and stop of PCNAdependent DNA replication, would play a major role
in p53's activities to ensure repair of DNA lesions, one
would have to postulate that waf1 nullizygote mice
should display a similar phenotype regarding tumor
predisposition as p53 null mice. However, this is not
the case, despite the ®nding that cells from waf1
nullizygote mice have largely lost the ability to arrest in
G1 after DNA damage (Deng et al., 1995). Therefore,
we assume that some other function of p53 is
preventing the accumulation of mutations in these
mice. We propose that such a function could be the
`basic function' of p53, preventing the accumulation of
mutations by its involvement in the repair of
endogenous DNA damage.
With the exception of sequence-speci®c transactivation of p53 target genes, which seems to be dependent
on post-translational modi®cation of p53 after DNA
damage, all of its other activities outlined above could,
in principle, be exerted also by a non-induced p53.
Possible limitations would only arise from the low
amounts of p53 usually present in normal cells.
However, even this argument is questionable, as it
has been demonstrated that the transactivation
function of p53 can be induced without a detectable
rise in p53 levels (Hupp et al., 1995). Although there is
no in vivo evidence for such a `basic' function of p53,
comparative analysis of the major biochemical activities exerted by the p53 core domain, sequence-speci®c
DNA binding and 3'-5' exonuclease activity (Figure 1),
prompted us to suggest a direct participation of p53 in
the repair of endogenous DNA damage, in preventing
faulty DNA repair, and possibly also in DNA
replication.
As it seems rather unlikely that p53 will act as an
exonuclease and simultaneously bind to DNA in a
sequence-speci®c manner, the localization of the 3'-45'
exonuclease activity of p53 to the same domain that
exerts sequence-speci®c DNA binding poses the
problem of how these activities are regulated. During
deletion mapping analyses we found that C-terminal
truncation of the C-terminal thirty amino acids of the
p53 molecule activated its exonuclease activity by
about a factor of ten, indicating that the C-terminal
regulatory domain of p53 negatively in¯uences the p53
exonuclease activity. The same, however, has been
shown for the sequence-speci®c DNA binding activity
Dual role model for p53 function
N Albrechtsen et al
of p53, underscoring the importance of the p53 Cterminus for the regulation of p53 functions. Yet,
whereas both activities were negatively regulated by the
p53 C-terminus, treatments which a€ected the regulation of p53 activities by the p53 C-terminus had
opposing e€ects on sequence-speci®c DNA binding and
the exonuclease activity of p53: treatments, which
activated sequence-speci®c DNA binding of p53, like
addition of the monoclonal antibody PAb421 which
binds to an epitope within the C-terminal regulatory
domain, or enhancing the phosphorylation status of
p53, strongly inhibited the p53 exonuclease activity
(Janus et al., 1999). This ®nding points to the exciting
possibility that p53 can exist in at least two di€erent
functionally active states. Since activation of sequencespeci®c DNA binding is considered to be a hallmark of
p53 activation after DNA damage, we conclude that
p53 looses its exonuclease activity when the sequencespeci®c DNA binding of p53 becomes activated after
DNA damage. Conversely, however, one can also
conclude that non-activated p53 exerts exonuclease
activity, implying that a non-activated p53 is not equal
to a non-functional one.
Possible functions of the p53 exonuclease activity
What could be the role of the p53 exonuclease activity
in DNA repair? Exonucleases are required for nearly
all processes of DNA metabolism, such as DNA
replication, long patch DNA repair, postreplicative
mismatch repair, and DNA recombination. An
important type of error avoidance mechanism requiring exonuclease activities is the mismatch repair
pathway. In bacteria this system invokes the coordinated action of the MutSLH damage recognition/
endonuclease complex along with the UvrD helicase,
DNA polymerase III, DNA ligase, single-strand DNA
binding protein, and any one of the exonucleases
ExoI(3'-45' exo), ExoVII (3'-45' and 5'-43' exo) or
RecJ (5'-43' exo) (Modrich, 1987, 1989). An involvement of p53's exonuclease in the postreplicative
mismatch repair pathway would be of particular
interest because this would link one of the enzymological functions of p53, i.e., its exonuclease activity, to
the tumor suppressor function of the mammalian
MutSLH homologues hMSH2, hMLH1, hPMS1, and
hPMS2. A role complementary to these mismatch
recognition proteins has already been proposed for p53
in mismatch recognition during recombination events
(see above). Functional loss of either of these proteins
leads to an increased incidence of colon carcinomas
(for reviews see Bodmer et al., 1994; Modrich, 1994;
Radman and Wagner, 1993). Furthermore, mutations
in any of these genes display a generalized increase in
spontaneous mutation rates, a replication error positive
(RER+) phenotype, and a resistance to alkylating
agents (Bhattacharyya et al., 1994; Leach et al., 1993;
Parsons et al., 1993). In this respect, it might be more
than coincidence that p53-de®cient animals show a
relative resistance to alkylating agents (Harvey et al.,
1993). Moreover, there is an inverse correlation
between RER+ status and p53 mutation in colorectal
cancer cell lines (Cottu et al., 1996), pointing to a
synergistic action of gene products involved in
mismatch repair with p53. This synergism is further
corroborated by the ®nding that male mice nullizygous
for both Msh2 and p53 develop tumors signi®cantly
earlier than either of the single mutants (Cranston et
al., 1997). Furthermore, there is some evidence that,
like the p53 protein itself (see above), an intact
mammalian MutSHL system displays an `anti-recombinogenic' e€ect (Fishel and Kolodner, 1995). In line
with the possibility of a direct involvement of p53 in
mismatch repair is the recent demonstration that wt
p53 can `sense' DNA mismatch lesions by tightly
binding to DNA containing insertion/deletion mismatches (Lee et al., 1995).
Another possibility is p53's involvement in error
avoidance by contributing a certain type of `proofreading' activity. There are at least six di€erent cellular
DNA polymerases designated as DNA polymerases a,
b, g, d, e, and z that are involved in di€erent aspects of
DNA synthesis. Two of these, namely DNA polymerases a and b, are devoid of a 3'-45' exonuclease
activity that excises mismatched nucleotides immediately after the incorporation step. Therefore, DNA
polymerases a and b are particularly error prone and
until now it is not known, how mismatched nucleotides
incorporated by these two polymerases are removed
(Kunkel, 1992). Moreover, both polymerases have a
strong tendency to introduce frame-shift mutations by
misinsertion or deletion of nucleotides (Huang, 1998).
Since p53 recognizes DNA bulges caused by insertion/
deletion mismatches (Lee et al., 1995), it might be
particularly suited to excise them via its 3'-45'
exonuclease. Last but not least, p53 might act as a
direct proofreader for the proofreader de®cient DNA
polymerases a and b.
A model for p53 function in the maintenance of genetic
integrity by DNA repair
Di€erent subclasses of p53 may perform di€erent
functions within the same cell
An important aspect for evaluating the activities of p53
in its induced and in its non-induced state is that not
necessarily all p53 molecules must be in the same
functionally active state, i.e. must exert the same
function. Functional heterogeneity of multifunctional
regulatory proteins is well known. Probably one of the
best documented examples is the SV40 T-Ag, the major
regulatory protein of SV40. Analyses of its subcellular
localization and of its biochemical activities in SV40
lytically infected cells clearly demonstrated the coexistence of several functionally di€erent subclasses of TAg molecules, involved in various aspects of viral
replication (reviewed in Deppert and Schirmbeck,
1995). Assuming a similar situation for the multifunctional p53 molecule, one could envision that even
after DNA damage only a certain fraction of the p53
molecules becomes activated for sequence-speci®c
transcription, whereas other molecules remain in their
non-induced state. Speci®city for selective activation
(or non-activation) of p53 subclasses can be provided
by subcellular compartmentalization of the p53 protein
and/or by its association with di€erent cellular
proteins. The existence of functionally di€erent
subclasses of p53 within the same cell so far has not
been proven. However, previous experiments from this
laboratory at least demonstrated the presence of p53 in
7713
Dual role model for p53 function
N Albrechtsen et al
7714
di€erent nuclear compartments (Deppert and Haug,
1986). Therefore, it is possible that the p53 population
not engaged in transcriptional regulation could exert
functions other than induction of growth arrest or
apoptosis, and directly participate in processes of DNA
repair via its various biochemical activities described
above. The exonuclease activity, for example, could be
involved in repair processes such as DSB repair, which
is thought to require DNA helicases, like the Ku
autoantigen (Suwa et al., 1994; Tuteja et al., 1994), and
exonucleases. Furthermore, p53 might act as an
external proofreader for errors produced by cellular
DNA polymerases involved in DNA replication and
DNA repair also under DNA damage conditions.
DNA polymerase a is certainly involved in nuclear
DNA replication (Wang, 1991), but also has some
function in DNA repair (Saxena et al., 1990; Oda et
al., 1996; Holmes and Haber, 1999), whereas DNA
polymerase b has been solely assigned to DNA repair
processes (Wood and Shivji, 1997). When DNA
damage has occurred, there is an apparent shut-o€ of
PCNA-dependent DNA replication (Wood and Shivji,
1997; Flores-Rozas et al., 1994). Although PCNAdependent DNA-repair synthesis still might continue
under conditions of p21waf1 induction (Li et al., 1994a,
see above), DNA-repair synthesis could also be
performed by the PCNA-independent and proofreader-free DNA polymerases a and/or b, for which p53
could provide the proof-reader function. If one
assumes that after damage di€erent functional subclasses of p53 will exist within the same cell, then the
ensuing increase of p53 protein levels not only will
activate the potential of p53 to transcribe p53 target
genes, leading to growth arrest and to shut-o€ of
PCNA-dependent DNA replication, but will also
increase the amount of p53 with a 3'-45' proofreading
exonuclease activity. Such p53 then could enhance the
accuracy of DNA repair synthesis performed by the
more error-prone DNA polymerases a and b.
The `dual role' model for p53 The major conclusion
which can be drawn from the above considerations is
that the current picture of p53 as a guardian of the
genome solely activated by cellular stress and/or DNA
damage will be an oversimpli®cation. We assume that a
non-induced p53 is not a protein without function. p53
in its non-induced state at least performs exonuclease
activity, which could be involved in a variety of
possible functions of p53 in DNA repair which all
contribute to avoid mutations in the genome, as
outlined above. We consider that the basic function
of p53 is the repair of endogenous DNA damage, and
the prevention of mutational events resulting from such
damage. Superimposed are the up to now better
characterized functions of p53 as a superior control
element in integrating cellular stress signals, followed
by the induction of either a growth arrest or of
apoptosis. This model of a dual role of p53 as a
guardian of the genome is outlined in Figure 2. If
induction of p53 will activate only a fraction of p53 for
sequence-speci®c transcription, the `basic function' of
p53 must not be restricted to p53 in its non-induced
state, and other, non-induced subclasses of p53 could
directly engage in the various repair processes outlined
Figure 2 Dual role of p53 as guardian of the genome. The current view of p53 as a superior control element in the responses to
various types of cellular stress is depicted in the right half of the ®gure (adapted from Hall et al., 1996). The left half provides an
extension of this model, according to which a non-induced p53 actively participates in the prevention of mutations arising from
endogenous DNA damage. Note that functions of a non-induced p53 can also operate under conditions of cellular stress, if one
assumes that after cellular stress di€erent functional subclasses can exist within the same cell (for details see text)
Dual role model for p53 function
N Albrechtsen et al
above. Thereby p53, after DNA damage then would be
able to exert its full range of possible biochemical
activities.
Clearly, this model is highly speculative, but testable.
For example, the postulated proofreader activity of p53
predicts a functional association of an exonucleaseactive p53 with the proofreader de®cient polymerases a
and/or û. Biosensor studies indeed suggest that p53
binds to both polymerases with considerable anity
(KuÈhn et al., 1998), and a speci®c complex between
p53 and an immunologically distinct subclass of
polymerase a could be detected in vivo (I Dornreiter,
unpublished observation). Moreover, pol a can be
anity-puri®ed together with p53 from monoclonal
antibody columns directed against pol a (F Grosse,
unpublished observations). Furthermore, Huang (1998)
recently showed that wt p53, but not mutant p53,
speci®cally enhanced the replication ®delity of polymerase a in an in vitro replication assay, strongly
supporting the idea that p53 can act as an exogenous
proofreader for this replicase. However, further
experimentation is necessary to con®rm and extend
these initial ®ndings.
Acknowledgments
Work from this laboratory was supported by the Dr
Mildred Scheel Stiftung (Deutsche Krebshilfe), the
Deutsche Forschungsgemeinschaft (DFG) and the Fonds
der Chemischen Industrie. The Heinrich-Pette-Institut is
®nancially supported by the Freie und Hansestadt Hamburg and by the Bundesministerium fuÈr Gesundheit. Parts
of this review have been published in Janus et al., CMLS,
55 (1999), 12 ±27.
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