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Oncogene (2003) 22, 233–245
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p53-associated 30 -50 exonuclease activity in nuclear and cytoplasmic
compartments of cells
Lilling Gila1, Novitsky Elena2, Sidi Yechezkel1 and Bakhanashvili Mary*,2
1
Department of Medicine C and Laboratory of Experimental Chemotherapy, Chaim Sheba Medical Center, Tel Hashomer 52621,
Israel; 2Infectious Diseases Unit, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel
The tumor suppressor protein p53 plays an important role
in maintenance of the genomic integrity of cells. p53
possesses an intrinsic 30 -50 exonuclease activity. p53 was
found in the nucleus and in the cytoplasm of the cell. In
order to evaluate the subcellular location and extent of
p53-associated 30 - 50 exonuclease activity, we established an in vitro experimental system of cell lines with
different nuclear/cytoplasmic distribution of p53. Nuclear
and cytoplasmic extracts obtained from LCC2 cells
(expressing a high level of cytoplasmic wild-type p53),
MCF-7 cells (expressing a high level of wild-type nuclear
p53), MDA cells (expressing mutant p53) and H1299 cells
(p53-null) were subjected to the analysis of exonuclease
activity. Interestingly, 30 -50 exonuclease was predominantly cytoplasmic; the nuclear extracts derived from all
cell lines tested, exerted a low level of exonuclease
activity. Cytoplasmic extracts of LCC2 cells, with a high
level of wild-type p53, showed an enhanced exonuclease
activity in comparison to those expressing either a low
level of wild-type p53 (in MCF-7 cells) or the mutant p53
(in MDA cells). Evidence that exonuclease function
detected in cytoplasmic extracts is attributed to the p53
is supported by several facts: First, this activity closely
parallels with levels and status of endogenous cytoplasmic
p53. Second, immunoprecipitation of p53 from cytoplasmic extracts of LCC2 cells markedly reduced the
exonuclease activity. Third, the observed 30 -50 exonuclease in cytoplasmic fraction of LCC2 cells displays
identical biochemical properties characteristic of recombinant wild-type p53. The biochemical functions include:
(a) substrate specificity; exonuclease hydrolyzes singlestranded DNA in preference to double-stranded DNA and
RNA/DNA template–primers, (b) efficient excision of 30 terminal mispairs from DNA/DNA and RNA/DNA
substrates, (c) the preferential excision of purine–purine
mispairs over purine–pyrimidine mispairs and (d) functional interaction with exonuclease-deficient DNA polymerase, for example, murine leukemia virus reverse
transcriptase (representing a relatively low fidelity enzyme), thus enhancing the fidelity of DNA synthesis by
excision of mismatched nucleotides from the nascent DNA
*Correspondence: M Bakhanashvili, Infectious Diseases Unit, Chaim
Sheba Medical Center, Tel Hashomer 52621, Israel,
E-mail: [email protected]
Received 22 May 2002; revised 2 October 2002; accepted 4
October 2002
strand. Taken together, the data demonstrate that wildtype p53 in cytoplasm, in its noninduced state, is
functional; it displays intrinsic 30 -50 exonuclease activity. The possible role of p53-associated 30 -50 exonuclease
activity in DNA repair in nucleus and cytoplasm is
discussed.
Oncogene (2003) 22, 233–245. doi:10.1038/sj.onc.1206111
Keywords: p53; 30 -50 exonuclease; cytoplasmic p53;
nuclear p53; DNA replication fidelity
Introduction
The tumor suppressor protein p53, a potent mediator of
cellular responses to DNA damage, is biochemically
involved in the induction of cell-cycle arrest, apoptosis
or DNA repair, all of which contribute to maintenance
of genetic stability in mammalian cells (Albrechtsen
et al., 1999; Prives and Hall, 1999). Under normal
conditions of cell growth, p53 exists at low levels in a
latent state (Soussi, 1995). However, various intracellular and extracellular signals, including ionizing radiation (Kastan et al., 1991), UV radiation (Nelson and
Kastan, 1994), hypoxia (Graeber et al., 1994) or
nucleotide deprivation (Linke et al., 1996), can induce
the formation of activated p53. The high levels of
functional p53 correlate with arrest at the G1 phase of
the cell cycle, allowing DNA repair prior to cell division
or elimination of cells with extensive DNA damage by
apoptosis (Cox et al., 1995). p53 is a multifunctional
protein with multiple modifications and biochemical
properties. The cellular response to DNA damage
presumably involves the capacity of p53 to bind to
specific DNA sequences and to function as a transcription factor, which both transactivates and transrepresses
specific genes involved in controlling cell cycle and
apoptosis (Prives and Hall, 1999). The transactivationindependent functions of p53 include DNA repair
through nonsequence-specific DNA binding. Indeed,
p53 acts in nucleotide excision repair (NER) to eliminate
pyrimidine dimers caused by UV light (Hwang et al.,
1999) and in base excision repair (BER) caused by DNA
hydrolysis and alkylation (Offer et al., 1999; Zhou et al.,
2001). Hence, by sequence-specific and nonsequencespecific DNA binding, p53 as the ‘guardian of the
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
234
genome’ might regulate the cellular response to DNA
damage in concert with the DNA repair machinery in
order to select among multiple pathways toward DNA
repair and apoptosis. Interestingly, while sequencespecific transactivation of p53 target genes is dependent
on post-translational modification of p53 after DNA
damage, all other activities could be displayed by a
noninduced p53; for example, it may play a role in
ribosomal biogenesis (Marechal et al., 1994); it may
actively participate in BER (Offer et al., 1999).
Various biochemical activities of p53 related to direct
involvement in DNA repair are associated with nonsequence-specific binding to DNA (El-Deiry et al., 1992;
Foord et al., 1993): (a) p53 itself has the ability to
recognize and interact with several forms of DNA such
as single-stranded (ss) and double-stranded (ds) DNA
ends, DNA with insertion–deletion lesion mismatches
(Steinmeyer and Deppert, 1988; Kern et al., 1991; Lee
et al., 1995); (b) p53 promotes the reannealing and
strand transfer of short oligonucleotides (Bakalkin et al.,
1994; Oberosler et al., 1993); (c) p53 checks the fidelity
of homologous recombination processes by specific
mismatch recognition (Dudenhoffer et al., 1998; Janz
et al., 2002); and (d) wild-type (wt) p53 exhibits intrinsic
30 -50 exonuclease activity (Mummenbrauer et al., 1996;
Huang, 1998). Evidently, recombinant wtp53, by
associated 30 -50 exonuclease, is able to preferentially
remove mispaired nucleotides from 30 - of the DNA
strand and enhance the accuracy of cellular and viral
DNA synthesis. Indeed, it might act as an external
proofreader for errors introduced by exonucleasedeficient cellular DNA polymerase a (Huang, 1998),
DNA polymerase a-primase (Melle and Nasheuer, 2002)
and by retroviral murine leukemia virus (MLV)
(Bakhanashvili, 2001a) or human immunodeficiency
virus (HIV) reverse transcriptase (RT) (Bakhanashvili,
2001b). This activity provides a molecular basis for p53
involvement in DNA replication machinery complexes
where proofreading is necessary, thus contributing to
the maintenance of genomic stability. Notably, the
exonuclease activity of p53 must not be restricted to its
noninduced state, but might also be exerted by a
subclass of p53 after DNA damage when the protein is
able to display its full range of possible biochemical
activities (Albrechtsen et al., 1999).
p53 was found in the nucleus, the cytoplasm or both
compartments of the cell. p53 is retained in the
cytoplasm during part of the normal cell cycle. The
localization of p53 in the nucleus is essential for its
normal function in growth inhibition or induction of
apoptosis. Several studies have shown that wtp53 occurs
in cytoplasm in a subset of human tumor cells such as
breast cancers, colon cancers and neuroblastoma (Stenmark-Askmalm et al., 1994; Bosari et al., 1995; Moll
et al., 1995). Notably, besides structural mutation and
the functional inactivation of wtp53, cytoplasmic
sequestration of p53 in tumor cells (that do not have
mutated p53) was suggested to be an important
mechanism in abolishing p53 function and in tumorigenesis (Sun et al., 1992; Stenmark-Askmalm et al.,
1994). Interestingly, results from a number of studies
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have indicated that p53 plays a role in the control of
protein synthesis. Cytoplasmic p53 functions include
translational repression through binding to mRNAs or
complexing with MDM-2, L5 ribosomal protein and/or
5.8S rRNA (Fontoura et al., 1992; Marechal et al.,
1994). The translational control by p53 is the first
example indicating the functionality and significance of
the presence of p53 in cytoplasm.
The role of p53-associated 30 -50 exonuclease in
DNA repair machinery was observed in an in vitro
model system with purified recombinant p53 and in
whole cells (Mummenbrauer et al., 1996; Huang, 1998;
Skalski et al., 2000; Bakhanashvili, 2001a; Ballal et al.,
2002). Since p53 is expressed constitutively in the cell
and is distributed in the nucleus and cytoplasm, it was of
interest to evaluate the subcellular location and extent of
p53-associated 30 -50 exonuclease activity in cells. It was
important to obtain p53 protein in the wt conformation
and at a sufficient amount to examine its activity in an in
vitro assay. Hence, in our study we took advantage of
our recent observation that in MCF-7 human breast
cancer cell line, even without exogenous DNA damage,
wtp53 is mainly accumulated in the nucleus, whereas
LCC2 cells (subline of MCF-7) maintain p53 predominantly within the cytoplasm (Lilling et al., 2002). These
properties make these cells particularly suitable as an
experimental model system, since under p53 noninducible conditions, the absence of activated apoptotic
pathways permits the analysis of p53-associated exonuclease activity in nuclear and cytoplasmic extracts,
which requires relatively moderate levels of p53.
We found that the cytoplasmic fraction of LCC2
cells, expressing a high level of wtp53, exhibits 30 -50
exonuclease activity and displays biochemical properties
characteristic of recombinant p53. Furthermore, depletion of p53 by anti-p53 monoclonal antibodies abolished
the observed exonuclease activity. These data suggest
that p53 in cytoplasm, in its noninduced state, is
intrinsically functional.
Results
30 -50 exonuclease activity in nuclear and cytoplasmic
extracts
According to several studies, the onset of different p53dependent activities correlates with the levels, localization and status of p53 in the cells. The fact that wtp53
normally is short-lived and is maintained at low levels in
unstressed cells makes it difficult to use cellular extracts,
either nuclear or cytoplasmic obtained from growing
cells, as a suitable source for the analysis of p53associated activities. However, nonstressed tumor cells
express relatively high levels of wtp53. In our previous
studies, both the immunohistochemistry and Western
blot analyses showed that p53 was located in the nucleus
of MCF-7 cells and in the cytoplasm of LCC2 cells
(Lilling et al., 2002). Hence, the experimental model
system that we utilized included nonstressed human
breast cancer cells MCF-7, LCC2 (both carrying wtp53),
p53 in cytoplasm exhibits 30 -50 exonuclease activity
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235
MDA-MB-231 (harboring mutant p53) and p53-null
H1299 cells. We looked for a possible relationship
between p53 and exonuclease activity. To accomplish
this, the nuclear and cytoplasmic extracts of these cell
lines were analysed for p53 expression by Western blot
analysis and for exonuclease activity. Consistent with
the published results, there is a difference in nuclear/
cytoplasmic distribution of p53 in these model cell lines
(Lilling et al., 2002): p53 is located mainly in the nuclear
fraction of MCF-7 and MDA cells, in the cytoplasmic
fraction of the LCC2 cells and is absent in H1299 cells
(Figure 1a). These nuclear and cytoplasmic fractions
were tested for the removal of the 30 -terminal mispair by
exonuclease activity using dsDNA oligonucleotide containing A : A mispair at the 30 -terminus as substrate. The
exonucleolytic activity was detected by excision of the
30 –terminal nucleotide, resulting in conversion of the
16-mer 50 -end labeled oligonucleotide to 15-mer, 14-mer,
etc. with the correctly paired 30 -terminus (Figure 1b).
Evidently, no exonuclease activity was detected in both
compartments of H1299 cells. Interestingly, the results
obtained revealed the existence of variations in constitutive exonuclease activity between nucleus and cytoplasm in association with p53. The nuclear extracts
derived from all cell lines tested (LCC2 – expressing low
levels of wtp53, MCF7 – expressing high levels of wtp53
or MDA – with mutant p53) exert low levels of
exonuclease activity. Conversely, we noticed that
cytoplasmic extracts display higher 30 -50 exonuclease
activity than that induced by nuclear extracts. These
results were reproduced four times with separate
preparations of nuclear and cytoplasmic extracts. The
formation of fragments was quantified by phosphorimaging. It is apparent that cytoplasmic extracts of
Figure 1 Subcellular localization of p53 and 30 -50 exonuclease activity in cytoplasmic and nuclear protein extracts. (a) Analysis of
p53 levels in MCF-7, H1299, MDA-MB-231 (MDA) and LCC2 cells by Western blotting. Cells were grown, harvested and nuclear and
cytoplasmic fractions were obtained as described in the Materials and methods section. Equal protein samples (10 mg) from both
nuclear (N) and cytoplasmic (C) fractions of these cells were subjected to SDS–PAGE and p53 protein expression was detected by the
Do-1 anti-human p53 mAb. (b) The same samples were utilized for 30 -50 exonuclease activity. 30 -terminal nucleotide excision was
examined using 30 -terminal A : A mismatch containing DNA/DNA template/primer (set II). The reaction was started by addition of
cytoplasmic or nuclear fraction (3 mg). After 10-min incubation at 371C, 5 ml aliquots were withdrawn and analysed on 16%
polyacrylamide gel, as described in the Materials and methods section. The position of the 16-mer primer is indicated by an arrow. (c)
Controls for the subcellular fractionation. The distribution of the nuclear marker PCNA and the cytoplasmic marker b-tubulin was
analyzed by immunoblotting to ascertain the purity of each fraction (to characterize nuclear and cytoplasmic fractions, respectively).
Equal amounts (10 mg) of nuclear and cytoplasmic fractions of MCF-7 and LCC2 cells were tested for the appropriate antibodies
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p53 in cytoplasm exhibits 30 -50 exonuclease activity
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LCC2 cells, carrying high levels of wtp53, exhibit an
enhanced exonuclease activity (57%) in comparison to
those expressing either a lower level of p53 in MCF-7
(12%) or the mutant p53 in MDA cells. It should be
noted that the fractionation procedure was performed in
parallel with all cell lines used. The fact that MCF-7 cells,
in which p53 was expressed in the nucleus, had low levels
of exonuclease activity in cytoplasm indicates the likelihood that the observed exonuclease activity in LCC2
cells is directly linked to cytoplasmic p53 (detected by
immunohistochemistry – Lilling et al. (2002) – and
Western blot analysis – Figure 1a). Nevertheless, it was
important to rule out the possibility that p53 in cytoplasm
may be due to leakage of nuclear proteins. Consequently,
the completeness of the subcellular fractionation was
determined by monitoring the distribution of known
nuclear and cytoplasmic marker proteins in MCF-7 and
LCC2 cells. As is evident from Figure 1c, b-tubulin, a
commonly used cytoplasmic marker, was found exclusively in the cytoplasmic fraction, whereas PCNA was
predominantly nuclear. Hence, specific distribution of
markers for cytoplasm and nucleus, respectively, indicates
that the subcellular fractionation procedure was appropriate. Taken together, the experiments revealed an
interesting observation correlating the extent of exonuclease activity with the level and status of p53 in
cytoplasm but not in the nucleus.
The observed exonuclease activity from the cytoplasmic fraction of LCC2 cells removes the 30 -terminal
nucleotide as a function of time (increasing incubation
time resulted in increased abundance of the shorter
species) (Figure 2a) and in a dose-dependent manner
(exonuclease activity was proportional to the concentration of the cytoplasmic extract) (Figure 2b).
DNA polymerase g in mitochondria possesses 30 -50
exonuclease activity (Gray and Wong, 1992; Olson and
Kaguni, 1992). To exclude the possibility that observed
exonuclease activity in cytoplasm stems from mitochondrial DNA polymerase g, highly enriched mitochondrial
fractions (M) from LCC2 cells (comprising a very
small portion of p53) along with mitochondria-free
cytoplasmic (CMF) fractions (comprising a great portion of p53) (Figure 3a) were tested for exonuclease
activity. As depicted from Figure 3b, the exonuclease
activity in the mitochondria-free cytoplasmic fraction
was significantly higher than in the mitochondrial
extract. These results suggest that there is a positive
correlation between the p53 accumulation in the
mitochondria-free cytoplasmic fraction and 30 -5 h
exonuclease activity. Apparently, mitochondrial DNA
polymerase g is not responsible for the observed
exonuclease activity in the cytoplasmic fraction.
To further investigate the possible correlation between p53 and exonuclease activity, it was essential to
confirm that the detected exonuclease activity in the
cytoplasmic fraction of LCC2 cells is attributed to p53.
We immunodepleted the p53 from the cytoplasmic
extracts of LCC2 cells (expressing high levels of p53
and exonuclease activity) to examine whether it would
abolish this function of the protein. The results obtained
show that the depletion of p53 protein from the
cytoplasmic fraction following immunoprecipitation by
Do-1 anti-p53 antibody (Figure 4a, lane 2) is concomitant with a marked decrease in exonuclease activity
(Figure 4b, lane 2). Nonspecific immunoprecipitation by
the anti-horse IgG (H+L) did not affect either p53
expression or exonuclease activity (lane 3 in Figure 4a
and b, respectively). In parallel, exonuclease reactions
performed in the presence of the p53-specific antibody
PAb421 (Figure 4c, lane 2), but not the nonspecific antihorse IgG (H+L) antibody (Figure 4c, lane 3), revealed
the inhibition of the excision 30 -terminal mispair. Thus,
the inhibition of excision reaction was most probably
caused by abolishing the p53 associated exonuclease
activity.
Collectively, the data indicate a possible link between
the presence of p53 in the cytoplasmic fraction and 30 50 exonuclease activity.
Figure 2 Exonuclease activity in the cytoplasmic fraction of LCC2 cells. (a) The time course of 30 -terminal nucleotide excision by
exonuclease of the cytoplamic fraction of LCC2 cells was examined by incubation with the 30 -terminal A : A mismatch containing
DNA/DNA template/primer (set II) for the indicated times. The reaction was started by the addition of cytoplasmic fraction (0.5 mg).
After 1-, 2-, 5- and 10-min incubation at 371C, 5 ml aliquots were withdrawn and analysed on 16% polyacrylamide gel. (b) The
exonuclease activity was tested under standard exonuclease assay conditions by increasing the concentration of cytoplasmic protein
extract. The mixtures were incubated for 10 min. Exonuclease reactions were performed as described in the Materials and methods
section. The position of the 16-mer primer is indicated by an arrow
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p53 in cytoplasm exhibits 30 -50 exonuclease activity
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Figure 3 p53 expression and 30 -50 exonuclease activity in
mitochondrial and cytoplasmic extracts of LCC2 cells. (a) Analysis
of p53 levels by Western blotting was performed with protein
samples from mitochondria-enriched fraction (M), mitochondriafree cytoplasmic fraction (CMF) and total cytoplasmic fraction (C)
of LCC2 cells. Identical protein samples (10 mg each) of the
subcellular fractions were subjected to SDS–PAGE and the p53
protein level was detected as described in Figure 1. (b) The same
samples were utilized for 30 -50 exonuclease activity. 30 -terminal
nucleotide excision by exonuclease was examined by incubation
with each subcellular fraction (1 mg) as described in Figure 1. 30 terminal nucleotide excision was examined using 30 -terminal A : A
mismatch containing DNA/DNA template/primer (set II). The
position of the 16-mer primer is indicated by an arrow
Substrate specificity of cytoplasmic 30 -50 exonuclease
Recent studies revealed that recombinant wtp53 removes 30 -terminal nucleotides from various nucleic acid
substrates. The spectrum of substrates utilized by p53
includes ssDNA, DNA/DNA and RNA/DNA substrates (Huang, 1998; Skalski et al., 2000; Bakhanashvili, 2001b). We attempted to elucidate potential
substrates for exonuclease detected in the cytoplasmic
fraction of LCC2 cells. To test this, the 32P-labeled 16mer primer alone or hybridized to either complementary
DNA or RNA were used as ssDNA, dsDNA and RNA/
DNA substrates, respectively. The results obtained
demonstrate that 30 -50 exonuclease activity is able to
hydrolyze dNMPs from the 30 -terminus of all three
substrates (Figure 5). However, the cleavage of the
ssDNA is substantially higher than that of the DNA/
DNA or RNA/DNA template–primers, thus indicating
that the cytoplasmic exonuclease, similar to recombinant wtp53, preferentially recognized the ss character of
these substrates. It should be noted that the exonuclease
activity is not associated with DNA polymerase, since
oligonucleotide bands greater than 16 nucleotides in
length are not detected even though dNTPs are included
in the reactions (data not shown). The observed
substrate specificity indicates that the exonuclease could
function in several DNA repair pathways during DNAand RNA-dependent DNA polymerization reactions.
The 30 -terminal base excision capacity of 30 - 50
exonuclease in cytoplasmic extracts from LCC2 cells
was further analysed under nonpolymerization conditions with four DNA/DNA (set II) or RNA/DNA (set I)
template–primers containing either an incorrect A : A,
A : C, A : G or correct A : T base pair at 30 -termini. The
ability of 30 -50 exonuclease from the cytoplasmic
fraction of LCC2 cells to remove 30 -terminal mispairs
from DNA/DNA and RNA/DNA substrates is illustrated in Figure 6a. It is apparent that under
nonpolymerization conditions, the exonuclease excises
the 30 -terminal nucleotide from all primers tested,
although there are variations in the degradation pattern
of the different mispairs. The exonuclease exhibits a
preference for mismatched rather than matched bases
with both substrates. Indeed, with the DNA/DNA
template–primer, removal of the 30 -terminal mismatched
A : A or A : G nucleotide (19 and 43%, respectively) is
more efficient than that of a complementary terminal
base pair A : T (9%), indicating a specificity toward the
degradation of a mispaired primer end. The trend in the
order of a 30 -terminal mispair excision opposite a
template A residue with both DNA/DNA and RNA/
DNA template–primers is pur.pur>pur.pyr. The mispair excision specificity was further assessed with
separate four template–primer substrates of DNA/
DNA (set III) or RNA/DNA (set IV), containing 30 terminal mispaired nucleotides opposite a template G
residue. As shown in Figure 6b, exonuclease in the
cytoplasmic fraction of LCC2 cells is most reactive
again with purine–purine G : A and G : G mispairs (17
and 12%, respectively). The specificity of mispair
excision with both substrates is G : A>G : G>G : T.
Evidently, there is a difference in mispair excision
efficiency between the two sets of template–primers
utilized; namely, the mispairs opposite target A
(Figure 6a) were excised more efficiently compared to
those opposite target G (Figure 6b). This dissimilarity
probably stems from (i) the nature of the mispair and/or
(ii) the difference in nearest neighbors from a template
target site (GA- purines in Figure 6a and CT-pyrimidines in Figure 6b). Notably, the same type of
preformed mismatches were chosen in the same
sequence context that was previously employed with
recombinant wtp53 with both DNA/DNA and RNA/
DNA substrates (Bakhanashvili, 2001a,b). The mispair
237
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p53 in cytoplasm exhibits 30 -50 exonuclease activity
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Figure 4 p53 expression and 30 -50 exonuclease activity in p53-depleted cytoplasmic fraction of LCC2 cells. Cytoplasmic fractions of
LCC2 cells (lane 1) immunodepleted by the Do-1 anti-human p53 mAb (lane 2) or by anti-horse IgG (H+L) were assayed for p53
expression by Western blotting (a) and for 30 -50 exonuclease activity (b), as described in the Materials and methods section.
Exonuclease activity in the cytoplasmic fraction of LCC2 cells was tested by incubation of the reaction mixture in the absence (lane 1)
or presence of Do-1 anti-human p53 mAb (lane 2) or of anti-horse IgG (H+L) (lane 3) (c). The position of the 16-mer primer is
indicated by an arrow
excision pattern detected in the current study is
compatible with similar results obtained previously with
recombinant wtp53. Thus, the data suggest that the 30 50 exonuclease from the cytoplasmic fraction of LCC2
cells is active when first binding to a 30 -terminus and
prefers transversion mutations over transition mutations. The preference for excision of mispaired 30 -termini
suggests a role in exonucleolytic proofreading, generating the correctly base-paired 30 -termini necessary for
continued DNA synthesis.
Functional interaction between 30 -50 exonuclease from
the cytoplasmic fraction of LCC2 cells and exonucleasedeficient MLV RT
For DNA polymerases that lack an intrinsic proofreading activity, it is possible that excision could be
performed by a separate exonuclease. To determine
whether exonuclease in the cytoplasmic fraction could
reduce the replication errors induced by exonucleasedeficient polymerase, the fidelity of DNA synthesis by
MLV RT (lacking intrinsic exonuclease activity and
representing a relatively low-fidelity DNA polymerase)
was evaluated in the presence of the cytoplasmic extract
of LCC2 cells. We used an in vitro gel fidelity assay that
proved to be useful to identify the proofreading
stimulatory factors. This system allows the simultaneous
detection of both degradation (exonucleolysis) and
Oncogene
extension (polymerization) to investigate the contribution of exonuclease activity in correcting MLV RT
errors during the DNA synthesis. In general, proofreading 30 -50 exonuclease activity enhances in less
stable DNA regions, leading to a reduction in base
substitution error frequencies in AT- vs GC-rich
sequences (Wang et al., 2002). Hence, sequences were
chosen to provide an AT-environment from a template
target site to maximize proofreading. To assess the
cooperation of the exonuclease from the cytoplasmic
fraction of LCC2 cells and MLV RT, we carried out
two reactions indicative of the fidelity of DNA synthesis:
the misinsertion and mispair extension, during
both DNA-dependent DNA polymerization (DDDP)
and RNA-dependent DNA polymerization (RDDP)
reactions.
(a) The insertional fidelity of MLV RT was examined
with both substrates using running-start template–
primer. The reactions were performed at a fixed
concentration of 0.5 mm of dATP, allowing the extension of the 16-mer primer. The results of the primer
extension assays show that MLV RT displays misinsertion activity with both substrates, incorporating dATP
opposite the template G at site 10 of template DNA
(Figure 7, lane 1) or at sites 2009 and 2005 of template
RNA (Figure 7, lane 3) following the initial incorporation of two running-start A’s prior to reaching the first
template target site G. Notably, the presence of primer
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
239
Figure 5 Excision of the 30 -terminal nucleotide from various
nucleic acid substrates by 30 -50 exonuclease from the cytoplasmic
extract of LCC2 cells. The ssDNA (50 -ATTTCACA TCTGACTT30 DNA/DNA (with 30 -terminal A : A mispair – set II) and RNA/
DNA (with 30 -terminal A : A), mispair – set I) substrates were
incubated with the cytoplasmic fraction of LCC2 cells for 20 min
and reaction products were analysed by polyacrylamide gel
electrophoresis as described under the Materials and methods
section. The position of the 16 mer primer is indicated by an arrow
extension products longer than 19-mer suggests that the
creation of first mismatch (A : G) was followed by
elongation. This reflects the ability of the enzyme not
only to misinsert a wrong dATP (mutator phenotype),
but also to extend the newly formed mispair. Interestingly, the misincorporation of dATP was reduced with
both template–primers in the presence of cytoplasmic
extract of LCC2 cells (Figure 7, lanes 2 and 4).
Apparently, the 30 -50 exonuclease activity in cytoplasmic fraction affected the polymerase selectivity for base
substitution errors during both DDDP and RDDP
reactions; it substantially reduced the number of
mismatched nucleotides incorporated into DNA.
(b) The efficient extension of mismatched 30 -termini of
DNA in concert with the ability to introduce mispairs is
a major factor affecting the low accuracy of DNA
synthesis exhibited by different DNA polymerases
(Perrino et al., 1989; Bakhanashvili and Hizi, 1992a,
1993). Therefore, the 30 -50 exonuclease from the
cytoplasmic fraction of LCC2 cells was further assessed
for its influence on the mispair extension ability of MLV
RT. The slow mismatch extension capacity of the
enzyme could allow a separate exonuclease to compete
for mismatched primer termini. Hence, the mispair
extension capacity of MLV RT was evaluated in the
presence of the cytoplasmic fraction of LCC2 cells by
analysing the extension of DNA/DNA and RNA/DNA
template–primers containing A : A and A : C mispairs
(substrates that were efficiently extended by MLV RT)
(Bakhanashvili and Hizi, 1992a) and the next complementary nucleotide dATP (as the only dNTP present).
Under the assay conditions, the polymerase displayed a
characteristic mispair extension profile with both substrates. Indeed, MLV RT elongates the A : A and A : C
mispairs, accumulating the correct A : T 17- and 18residue primers (Figure 8, lanes 1, 2, 5 and 6). However,
the mispair extension pattern was different in the
presence of the cytoplasmic fraction of LCC2 cells;
namely, a reduction in elongation of the primer was
observed. Indeed, hydrolysis of the 30 -terminal nucleotide resulted in decreased intensities of the 18-mer and
17-mer bands, concomitant with the appearance of (15mer, 14-mer and etc.) products resulting from 30 -50
exonuclease activity (Figure 8, lanes 3, 4, 7 and 8).
Hence, the data indicate that MLV RT with both
template–primers displays a lower capacity of mispair
extension in the presence of the cytoplasmic fraction of
LCC2 cells expressing wtp53. These proofreading results
imply a functional cooperation of polymerization (by
MLV RT) and excision (by exonuclease from the
cytoplasmic fraction) activities, which act in a coordinated manner during DNA synthesis at the template–
primer site to achieve a high accuracy of DNA synthesis.
These findings suggest that the exonuclease in cytoplasm
might fulfill a proofreading function; namely, it can
correct replication errors made by exonuclease-deficient
DNA polymerase and its biochemical properties are
fully consistent with those of recombinant wtp53.
Discussion
Current studies revealed two interesting observations:
(a) p53 in the cytoplasmic fraction of nonstressed cells is
functional; it exerts 30 -50 exonuclease activity and
(b) p53 in nuclear extracts displays a relatively low level
of 30 -50 exonuclease activity in comparison to cytoplasmic extracts.
Using an in vitro model system, we found that the
cytoplasmic extracts of LCC2 cells expressing wtp53
exhibited a high level of 30 -50 exonuclease activity. The
following evidence allows us to attribute the detected
exonuclease activity to p53: (a) The observed 30 -50
Oncogene
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
240
Figure 6 Mispair excision by 30 -50 exonuclease from the cytoplasmic fraction of LCC2 cells. (a) Excision reactions for 30 -terminal
nucleotides were carried out with DNA/DNA and RNA/DNA template–primers containing the 30 -terminal mispaired nucleotide
opposite a template A residue (sets II and I, respectively). Lane A – template-primer with 30 -terminal A : A mispair. Lane C – template–
primer with 30 -terminal A : C mispair. Lanes G – template-primer with 30 -terminal A : G mispair. Lane T –template–primer with 30 terminal correct A : T base pair. (b) Excision reactions for 30 -terminal nucleotides were carried out with DNA/DNA and RNA/DNA
template–primers containing 30 -terminal nucleotides opposite a template G residue (sets IV and III, repectively). Lane A - templateprimer with 30 -terminal G:A mispair. Lane G – template–primer with 30 -terminal G:G mispair. Lanes T – template–primer with 30 terminal G:T mispair. Lane C – template–primer with 30 -terminal correct G : C base pair. The position of the 16-mer primer is indicated
by an arrow
exonuclease activity in cytoplasmic extracts closely
correlates with the endogenous levels and status of
p53. Indeed, cytoplasmic extracts of LCC2 cells expressing high levels of wtp53 showed an enhanced exonuclease activity in comparison to cytoplasmic extracts
expressing either low levels of wtp53 (in MCF7 cells) or
the mutant p53 with altered conformation (in MDA
cells). (b) This exonuclease activity was greatly reduced
in predepleted extracts with specific anti-p53 antibody.
(c) The 30 -50 exonuclease in the cytoplasmic fraction
from LCC2 cells exhibits identical biochemical functions
characteristic for recombinant wtp53: (1) It removes
30 -terminal nucleotides from various nucleic acid subOncogene
strates: ssDNA, DNA/DNA and RNA/DNA template–
primers. (2) It hydrolyzes dNMPs from ssDNA as the
optimal substrate. (3) It displays a marked preference
for excision of a mismatched vs correctly paired 30 terminus with both RNA/DNA and DNA/DNA
template–primers. (4) It exerts the preferential excision
of purine–purine (e.g. A : A, A : G, G : A, G : G) mispairs
over purine–pyrimidine (e.g. A : C, G : T) mispairs. (5) It
excises nucleotides from nucleic acid substrates independently from DNA polymerase. (6) It fulfills the
requirements for proofreading function; acts coordinately with the exonuclease-deficient DNA polymeraseMLV RT to enhance the fidelity of DNA synthesis by
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
241
Figure 7 Misincorporation by MLV RT with DNA/DNA and RNA/DNA template–primers in the presence of the cytoplasmic
fraction of LCC2 cells. The DNA/DNA and RNA/DNA template–primers were incubated with MLV RT and 0.5 mm dATP in the
absence (lanes 2 and 5) or presence of the cytoplasmic fraction of LCC2 cells (lanes 3 and 6). The positions of the primers (lanes 1 and
4) are indicated by arrows
Figure 8 Mispair extension by MLV RT with DNA/DNA and RNA/DNA template-primers in the presence of the cytoplasmic
fraction of LCC2 cells. The 30 -terminal mismatch containing DNA/DNA and RNA/DNA template–primers were incubated with MLV
RT and 0.5 mm dATP in the absence (lanes 1, 2, 5 and 6) or presence of the cytoplasmic fraction of LCC2 cells (lanes 3, 4, 7 and 8).
Lane A – template–primer with 30 -terminal A : A mispair. Lane C – template–primer with 30 -terminal A : C mispair. The position of the
16-mer primer is indicated by an arrow
excision of mismatched nucleotides from the nascent
DNA strand with both RNA/DNA and DNA/DNA
template–primers (Mummenbrauer et al., 1996; Huang,
1998; Skalski et al., 2000; Bakhanashvili, 2001a, b).
Notably, the purity of cellular fractionation was
determined by monitoring the distribution of proteins
that have specific cellular localization (Figure 1c). Thus,
our results appear to be an accurate reflection of the
Oncogene
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
242
presence of p53 in the cytoplasm. Collectively, the data
indicate that wtp53 in cytoplasm, in its noninduced
state, displays intrinsic 30 -50 exonuclease activity.
Interestingly, 30 -50 exonuclease activity and sequencespecific DNA binding are mediated by the p53 core
domain. Evidently, these two activities are mutually
exclusive activities; they are subject to different and
opposing regulatory mechanisms. It was suggested that
p53 loses its exonuclease activity when the sequencespecific DNA binding of p53 becomes activated (Janus
et al., 1999). Importantly, cytoplasmic p53 fails to bind
to its specific DNA target (Ostermeyer et al., 1996). This
property is consistent with our results that p53 in
cytoplasm may act as exonuclease. It is noteworthy, that
in nonstressed cells p53 exists in a transcriptional inert
state. In this form, p53 may participate in DNA repair
pathways. The fact that purified recombinant p53 as
well as p53 in cytoplasmic extract exerts 30 -50
exonuclease activity suggests that this function probably
does not depend on p53 transactivation activity.
The biochemical difference between the p53 in nuclear
and cytoplasmic compartments raises questions as to
whether nuclear p53 loses exonuclease function of
cytoplasmic p53 or acquires an additional function
(e.g. efficient sequence-specific DNA binding and
transactivation). Our fractionation procedure consists
of nuclear protein extraction in the presence of 420 mm
NaCl (see the Materials and methods section). It was
demonstrated that increased ionic strength destabilizes
and dissociates binding of p53 to DNA (Butcher et al.,
1994). Hence, the possibility that p53-associated 30 -50
exonuclease activity is easier detected in cytoplasm
(when it is not bound to DNA) than in the nucleus
is highly unlikely. There is a possibility that the difference stems from one of the following mechanisms:
(i) intramolecular (e.g. post-translational modification,
conformational) or (ii) intermolecular (binding to
another protein). (i) Post-translational modifications
(e.g. phosphorylation, acetylation) play a critical role in
the outcome of protein activation (Meek, 1998). Hence,
it will be interesting to examine whether any of the posttranslational modifications are involved in regulating
the ability of p53 to serve as an exonuclease in the
nucleus and in the cytoplasm. wt p53 can exist in two
different conformations; mutant and wt. Changes in the
conformational status of endogenous p53 have been
reported in cultured cells, and suggested that this may be
an important process in its functional activity (Milner
and Watson, 1990; Sabapathy et al., 1997). Recently, it
was demonstrated that nuclear translocation of p53 can
result in a change in the conformation from mutant (in
cytoplasm) to wild type (in nucleus) (Gaitonde et al.,
2000). Hence, it is conceivable that the alteration of p53
protein conformation is responsible for the low level of
exonuclease activity in the nucleus. (ii) The interaction
of p53 with other proteins has key roles in its various
cellular activities. There is a possibility that the
selectivity of the functions can be provided by its
association with different DNA repair proteins.
Furthermore, recent studies demonstrate that p53 has
the capacity for simultaneous binding of both F-actin
Oncogene
and DNA containing insertion-deletion mismatches
(Okorokov et al., 2002). It is conceivable to speculate
that in the nucleus, upon interaction with the F-actin
and DNA repair proteins, p53 becomes modified and is
then liberated to exert various functions. It is noteworthy that changes in the conformation of p53 may be
mediated also by association with other proteins.
In the nucleus, cells developed multiple DNA repair
pathways to protect themselves from different types of
endogenous and exogenous DNA damage. p53-dependent BER repair occurs in vivo and does not depend on
p53 transactivation activity (Offer et al., 1999). There is
a possibility that in non-stressed cells, in an endogenous
DNA damage status, p53 in the nucleus is more
competent for the p53-associated BER pathway than
for 30 -50 exonuclease activity. Importantly, human
p53, which is induced by high doses of g irradiation,
exhibits approximately 10-fold more exonuclease activity, thus indicating that this biochemical function of p53
may be subject to stimulation by exogenous stimuli
(Huang, 1998). Hence, it is of interest to elucidate 30 -50
exonuclease activity in the nucleus and cytoplasm of the
cells with activated p53 induced by drug treatments
(in the absence of DNA damage) or following UV
irradiation (in the presence of DNA damage). The
studies are presently under way. In this regard, it is
attractive to speculate that the induction allows p53
to function in several ways: as a regulator of transcription, as a facilitator of BER and as an exonucleolytic
proofreader. Moreover, there is a possibility that both
transcription-independent pathways act in synergy,
thereby amplifying the potency of involvement of p53
in DNA repair.
There are several reports describing exonucleases not
associated with DNA polymerases. The exonucleases
detected were purified and characterized from whole
cells or from nuclear extracts of human and animal cells
(Vazquez-Padua et al., 1990; Harrington et al., 1993;
Perrino et al., 1994; Hoss et al., 1999). However, only
one has been identified in the cytoplasm of human H9
cells (Skalski et al., 1995). The exonuclease activity
detected in the cytoplasm of LCC2 cells and the
cytosolic exonuclease from H9 cells exhibit some similar
properties: (1) divalent metal requirement and salt
profile (data not shown) and (2) substrate specificity;
namely, both exonucleases are reactive with ssDNA,
DNA/DNA and RNA/DNA substrates. However, there
is a difference in mispair excision specificity. The
cytosolic exonuclease from H9 cells was most reactive
with the purine–pyrimidine mispair (e.g. G:T), while
exonuclease, either from the cytoplasmic extract of
LCC2 cells or associated with recombinant p53
(Bakhanashvili, 2001b), prefers purine–purine mispairs
(e.g. A:A, A:G, G:G, G:A) over the purine-pyrimidine
mispairs (e.g. A:C, G:T). Thus, the behavior of the
exonuclease from the cytoplasmic fraction of LCC2 cells
on mismatch-terminated DNA indicates that probably it
is a previously uncharacterized exonuclease activity.
Different functional subclasses of p53 exist within the
same cell that may perform different functions according to their subcellular localization. p53 is retained in the
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
243
cytoplasm during part of the normal cell cycle as well as
in certain tumor cells. It was suggested that p53 might be
associated with cytoplasmic ribosomes and that the p535.8S rRNA complex might be involved in ribosome
biogenesis and/or translational regulation in the cell
(Fontoura et al., 1992). These data, taken together with
our results that p53 in the cytoplasm may serve as an
exonucleolytic proofreader, indicate that noninduced
p53 is functional and is able to elicit a spectrum of
different biological effective pathways in the cytoplasm
as well as in the nucleus. It is remarkable that although
p53 displays various activities, not all functions are
active simultaneously. Hence, these observations raise
the question as to whether p53 may serve a dual purpose
in cytoplasm, i.e. translation control and exonucleolytic
proofreading, independently or simultaneously.
A particular 30 -50 exonuclease might have more than
one function in the cell (Shevelev and Hubscher, 2002).
The data obtained raise questions about the possible
functional role of p53-associated 30 -50 exonuclease
activity in the cytoplasm of normal and tumor cells. In
unstressed cells, this activity of p53 may be viewed as a
‘housekeeping’ function. Eukaryotic RNA species require exonucleolytic activities for processing and for
their turnover, which may have an impact on the extent
and regulation of translation (Wickens et al., 1997).
Thus, there is a possibility that p53 by associated
exonuclease activity may be involved in the efficiency
and fidelity of protein synthesis. The fact that the
exonuclease function of p53 may be subject to stimulation by exogenous stimuli raises the possibility that the
induction allows p53 to function in two ways: (a) as an
exonuclease; p53 could provide the cell with a mechanism for recognizing and destroying ssDNA of pathogen
and (b) as a proofreader for DNA polymerases lacking
inherent exonuclease. Viruses use cell proteins for
multiple purposes during their intracellular replication
cycles. p53 protein by its ability to excise mispaired
bases from DNA/DNA and RNA/DNA template–
primers may be relevant to the fidelity of DNA
replication of viruses in cytoplasm, e.g. hepatitis B virus
(HBV) or HIV. In our previous studies, we noticed an
enhancement of the fidelity of HIV-1 RT by recombinant p53 (Bakhanashvili, 2001b). Notably, HIV infections seem to increase the intracellular level of p53
(Genini et al., 2001). In view of these observations, it
seems reasonable to hypothesize that p53 in the
cytoplasm may provide a proofreading function for
HIV-1 RT (manuscript in preparation). The observations presented here open new avenues for investigating
the role of p53-associated exonuclease in cytoplasm.
1640+10% FCS. All lines were maintained in a humidified
95% air, 5% CO2 atmosphere.
Subcellular fractionation and Western blotting
Cells were harvested by trypsin-EDTA, washed by PBS and
fractionated according to Andrews and Faller (1991). Incubation was performed in the presence of ice-cold hypotonic
buffer (10 mm HEPES-KOH pH 7.9, 1.5 mm MgCl2, 10 mm
KCl, O.5 mm DTT, 0.2 mm PMSF) and 0.6% of NP-40 for
15 min followed by centrifugation at 15 000 g for 2 min at 41C,
and the supernatant was the cytoplasmic fraction. The pellet
was washed once with the hypotonic buffer. Nuclear proteins
were extracted by incubation on a rotating platform for 20 min
in hypertonic buffer (20 mm HEPES-KOH, pH 7.9. 25%
glycerol, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm
DTT, 0.2 mm PMSF) followed by centrifugation at 15 000 g for
15 min at 41C and the supernatant was the nuclear fraction.
Protein concentration from both fractions was determined
using the Bradford assay (Bio-Rad Lab., Hercules, CA, USA).
Identical protein amounts of both fractions were subjected to
PAGE. The p53 protein was detected by the Do-1, anti-p53
monoclonal antibody (mAb) (ascitic fluid diluted in PBST (a
generous gift from Prof. V. Rotter, Weizmann Inst., Israel)
1:300), followed by HRP-conjugated goat-anti-mouse Ab
(Jackson Lab., West Grove, PA, USA). Visualization of the
p53 expression was performed by an EzECL kit (Biological
Industries, Bet Haemek, Israel) according to the protocol.
b-tubulin was detected by anti-human b-tubulin mAb and
PCNA by anti-human PCNA mAb (purchased from Zymed
Lab. Inc. So, San Francisco, CA, USA).
The cytoplasmic fraction of LCC2 cells was incubated in the presence of Do-1,
anti-human p53 monoclonal antibody (mAb) or with antihorse IgG (H+L) mAb with 360o rotation at 41C, followed
by incubation in the presence of anti-mouse IgG conjugated to agarose (Sigma). The supernatants were collected after removal by centrifugation at 14 000 g for 5 min
at 251C and examined for p53 expression by Western
Blotting and exonuclease reactions.
Immunoprecipitation for p53 protein
Mitochondria were
prepared as described (Bogenhagen and Clayton, 1974).
Briefly, cells washed in TD buffer were centrifuged (300g at
41C) and resuspended in MgRSB buffer (1.5 mm MgCl2,
10 mm NaCl, 10 mM Tris, pH 7.5) and incubated for
10 min. Swollen cells were disrupted in a glass Dounce
homogenizer to yield approximately 95% of free nuclei.
Mitochondria was pelleted from the washed homogenate,
resuspended in manitol–sucrose buffer and layered over a
1–2 m discontinuous sucrose gradient and spun at 41C for
30 min at 110 000 r.p.m. The mitochondria (M) were
collected from the 1.0–1.5 sucrose interphase. The supernatant was referred to as the mitochondria-free cytoplasmic
fraction (CMF).
Preparation of mitochondrial fraction
Materials and methods
Cell lines and culture medium
The MCF-7 and MDA-MB-231 human breast cancer cell lines
were grown in DMEM+10% fetal calf serum (FCS). The
LCC2 cells (subclone derived from MCF-7) were grown in
DMEM lacking phenol red+10% charcoal stripped FCS
(CSS), H1299 cell line was grown in the presence of RPMI
Template–primers Four template–primer substrates were
used for the excision and polymerization reactions. The first
(set I), ribosomal RNA (rRNA), as a native substrate (a
mixture of 16S and 23S Eschericia coli rRNA), was primed
with 16-mer oligonucleotide primer, which hybridizes to the
nucleotides at positions 2112–2127 of the 16S rRNA. Four
versions of primers were synthesized separately. They are
Oncogene
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
244
identical except for the 30 -terminal nucleotide (N), which is
either A, C, G or T. The sequence of these oligonucleotide
primers is 50 -ATTTCACATCTGACTN-30 and (a). The
second (set II) is an (N)30 synthetic oligonucleotide DNA
template with a sequence derived from nucleotides 2100–
2129 of E. coli 16S rRNA (50 -AGGCGGTTTGTTAAGTCAGATGTGAAATCC-30 ). This DNA is primed with an
(N)16 oligonucleotide (utilized with rRNA template) that
hybridizes to the nucleotides at positions 13–28 of the
template DNA. Consequently, by hybridizing the primer
oligonucleotides to the rRNA template at position 2112 or
DNA template at position 13, the A : A, A : C, A : G
mispairs or the A:T correctly paired terminus were
produced. The third (set III) is the same (N)30 template
DNA primed with an (N)16 oligonucleotide 50 -GGATTTCACATCTGAN-30 (b) (N ¼ A, C, G or T), which
hybridizes to the nucleotides at positions 15–30. The fourth
(set IV) is composed of the rRNA primed with 16-mer
oligonucleotide (b), which hybridizes to the nucleotides at
positions 2142–2129 of the 16S rRNA. The sequence of the
template–primers used for the experiments of mispair
excision, misinsertion and mispair extension are depicted
in Figures 6–8. The primers were end-labeled at the 50 -end
with T4 polynucleotide kinase purchased from MBI
(Fermentas) and [g-32P]ATP. Unincorporated radioactivity
was removed by using G-25 microspin columns (Pharmacia
Biotech.), according to the instructions of the producer.
The end-labeled primers were annealed to the template
RNA or DNA as described (Bakhanashvili and Hizi,
1992b, 1993).
The 30 -50 exonucleolytic activity was
measured as the removal of 30 -terminal nucleotides (correct
or incorrect) from the 50 -[g-32P] end-labeled oligonucleotide,
determined by the increase in mobility during the gel
electrophoresis. The incubation mixture (12.5 ml) contained
50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 1 mm DTT, 0.1 mg/
ml BSA and 50 -end-labeled various substrates. The reaction
was started by the addition of 3 mg of nuclear or
cytoplasmic extract. The variables, including reaction time
and amounts of the extracts, are given in the legends to the
figures. Assays were carried out at 371C. At appropriate
time points the reactions were stopped by the addition of an
equal volume of formamide gel loading buffer, followed by
incubation at 901C for 3 min. The reaction products were
resolved by electrophoresis through a 16% polyacrylamide
sequencing gel (PAGE). Exonuclease activity, measured as
the reduction in size of labeled oligonucleotides, was
detected by autoradiography. The excision products were
quantified by densitometric scanning of gel autoradiographs.
To evaluate the
interaction between exonuclease in the cytoplasmic fraction
of the cells and MLV RT, the same template–primers (sets I
and II) used for the exonuclease activity were utilized as
substrates. When present, MLV RT was at 1.5 U/ml. For
site-specific nucleotide misinsertion, a correctly paired
template–primer substrates was used to analyse the dATP
misincorporation opposite the G residues at position 10 of
the DNA template or at position 2009 of the RNA
template. For the mispair extension, elongation of the 50 end-labeled template-primers was examined in the presence
of dATP as the only dNTP present. The dNTPs used were
of the highest purity available (Pharmacia Biotech.) with no
detectable traces of contamination by other dNTPs. The
reaction products were analysed by electrophoresis through
16% PAGE as described above. Degradation or extension
of the 50 -end-labeled primers was detected by autoradiography.
Exonuclease/polymerase coupled assays
Exonuclease assays
Acknowledgments
The authors thank Prof. E Rubinstein for critically reading the
manuscript and Kovalsky Victoria for excellent technical
assistance. This research was supported by a grant from Israel
Cancer Research Fund (ICRF) and by a grant from Israel
Cancer Association.
References
Albrechtsen N, Dornreiter I, Grosse F, Kim E, Wiesmuller L
and Deppert W. (1999). Oncogene, 18, 7708–7717.
Andrews NC and Faller DV. (1991). Nucleic Acid Res., 19,
2499.
Bakalkin G, Yakovleva T, Selivanova G, Magnusson KP,
Szekely L, Kiseleva E, Klein G, Terenius L and Wiman KG.
(1994). Proc. Natl. Acad. Sci. USA, 91, 413–417.
Bakhanashvili M. (2001a). Eur. J. Biochem., 268, 2047–2054.
Bakhanashvili M. (2001b). Oncogene, 20, 7635–7644.
Bakhanashvili M and Hizi A. (1992a). FEBS Lett., 306,
151–156.
Bakhanashvili M and Hizi A. (1992b). Biochemistry, 31,
9393–9398.
Bakhanashvili M and Hizi A. (1993). Biochemistry, 32,
7559–7567.
Ballal K, Zhang W, Mukhopadyay T and Huang P. (2002).
J. Mol. Med., 80, 25–32.
Bosari S, Viale G, Roncalli M, Graziani D, Borsani G, Lee
AK and Coggi G. (1995). Am. J. Pathol., 147, 790–798.
Oncogene
Bugenhagen D and Clayton DA. (1974). J. Biol. Chem., 249,
7991–7995.
Butcher S, Hainaut P and Milner J. (1994). Biochem. J., 298,
513–516.
Cox LS, Hupp T, Midgley CA and Lane DP. (1995). EMBO
J., 14, 2099–2105.
Dudenhoffer C, Rohaly G, Will K, Deppert W and
Wiesmuller L. (1998). Mol. Cell. Biol., 18, 5332–5342.
El-Deiry W, Kern SE, Pietenpol JA, Kinzler KW and
Vogelstein B. (1992). Nat. Genet., 1, 45–49.
Fontoura BMA, Sorokina EA, David E and Carroll RB.
(1992). Mol. Cell. Biol., 12, 5145–5151.
Foord O, Navot N and Rotter V. (1993). Molec. Cell. Biol., 13,
1378–1384.
Gaitonde SV, Riley JR, Qiao D and Martinez JD. (2000).
Oncogene, 19, 4042–4049.
Genini D, Sheeter D, Rought S, Zaunders JJ, Susin SA,
Kroemer G, Richman DD, Carson DA, Corbeil J and Leoni
LM. (2001). FASEB J., 15, 5–6.
p53 in cytoplasm exhibits 30 -50 exonuclease activity
G Lilling et al
245
Graeber T, Peterson J, Tsai M, Monica K, Fornace AJ and
Giaccia A. (1994). Mol. Cell. Biol., 14, 6264–6277.
Gray H and Wong TW. (1992). J. Biol. Chem., 267,
5835–5841.
Harrington JA, Reardon JE and Spector T. (1993). Antimicrob. Agents Chemother., 37, 18–20.
Hoss M, Robins P, Naven TJ, Pappin DJ, Sgouros J and
Lindhal T. (1999). EMBO J., 18, 3868–3875.
Huang P. (1998). Oncogene, 17, 261–270.
Hwang BJ, Ford JM, Hanawalt PC and Chu G. (1999). Proc.
Natl. Acad. Sci. USA, 96, 424–428.
Janus F, Albrechtsen N, Knippschhild U, Wiesmuller L,
Grosse F and Deppert W. (1999). Molec.Cell. Biol., 19,
2155–2168.
Janz C, Susse S, Wiesmuller L. (2002). Oncogene, 21, 2130–
2140.
Kastan MB, Onyekwere O, Sidransky D, Vogelstein B and
Craig RW. (1991). Cancer Res., 51, 6304–6311.
Kern SE, Kinzler KW, Baker SJ, Nigro JM, Rotter V, Levine
AJ, Friedman P, Prives C and Vogelstein B. (1991).
Oncogene, 6, 131–136.
Linke SP, Clarkin KC, Di Leonardo A, Tsou A and Wahl
GM. (1996). Genes Dev., 10, 934–947.
Lee S, Elenbaas B, Levine A and Griffith J. (1995). Cell, 81,
1013– 1020.
Lilling G, Nordenberg J, Rotter V, Goldfinger N, Peller S and
Sidi Y. (2002). Cancer Invest., 20, 509–517.
Marechal V, Elenbaas B, Piette J, Nicolas J and Levine AJ.
(1994). Mol. Cell. Biol., 14, 7414–7420.
Meek DW. (1998). Int. J. Radiat. Biol., 74, 729–737.
Melle C and Nasheuer H. (2002). Nucleic Acids Res., 30,
1493–1499.
Milner J and Watson JV. (1990). Oncogene, 5, 1683–1690.
Moll UM, LaOuglia M, Benard J and Riou G. (1995). Proc.
Natl. Acad. Sci. USA, 92, 4407–4411.
Mummenbrauer T, Janus F, Muller B, Wiesmuller L, Deppert
W and Grosse F. (1996). Cell, 85, 1089–1099.
Nelson W and Kastan MB. (1994). Mol. Cell Biol., 14,
1815–1823.
Oberosler P, Hloch P, Rammsperger U and Stahl H. (1993).
EMBO J., 12, 2389–2396.
Offer H, Wolkowicz R, Matas D, Blumenstein S, Livneh Z and
Rotter V. (1999). FEBS Lett., 450, 197–204.
Okorokov A, Rubbi CP, Metcalfe S and Milner J. (2002).
Oncogene, 21, 356–367.
Olson MW and Kaguni LS. (1992). J. Biol. Chem., 267,
23136–23142.
Ostermeyer AG, Runko E, Winkfield B, Ahn B and
Moll UM. (1996). Proc. Natl. Acad. Sci. USA, 93, 15190–
15194.
Perrino FW, Preston BD, Sandell LL and Loeb LA. (1989).
Proc. Natl. Acad. Sci. USA, 86, 8343–8347.
Perrino FW, Miller H and Ealey KA. (1994). Biol. Chem., 269,
16357–16363.
Prives C and Hall PA. (1999). J. Pathol., 187, 112–126.
Sabapathy K, Klemm M, Jaenisch R and Wagner EF. (1997).
EMBO J., 16, 6217–6229.
Skalski V, Liu S-H and Cheng Y-C. (1995). Biochem.
Pharmacol., 50, 815–821.
Skalski V, Lin Z, Choi BY and Brown KR. (2000). Oncogene,
19, 3321–3329.
Shevelev I and Hubscher U. (2002). Nat. Rev. Mol. Cell Biol.,
3, 1–12.
Soussi T. (1995). Molecular Genetics of Cancer. Cowell JK
(ed). Bios Scientific: Oxford, UK, pp. 135–178.
Steinmeyer K and Deppert W. (1988). Oncogene, 3,
501–507.
Stenmark-Askmalm M, Stal O, Sullivan S, Ferraud L, Sun
XF, Carstensen J and Nordenskjold B. (1994). Eur. J.
Cancer, 30A, 175–180.
Sun XF, Cartensen JM, Zhang H, Stal O, Wingren S,
Hatschek T and Nordenskjold B. (1992). Lancet, 340,
1369–1373.
Vazquez-Padua MA, Starnes MC and Cheng Y-C. (1990).
Cancer Commun., 2, 55–62.
Wang Z, Lazarov E, O’Donnell and Goodman MF. (2002).
J Biol. Chem., 277, 4446–4454.
Wickens M, Anderson P and Jackson RJ. (1997). Curr. Opin.
Genet. Dev., 7, 220–232.
Zhou J, Ahn J, Wilson SH and Prives C. (2001). EMBO J., 20,
914–923.
Oncogene