From the Departments of Radiation Oncology and
¶ Radiology, Montefiore Medical Center, Albert Einstein College of
Medicine, Bronx, New York 10467
Received for publication, October 11, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was
demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1
also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional
consequence of the HSP70/HAP1 interaction was a 10-100-fold
enhancement of endonuclease activity at abasic sites. The physical and
functional interaction between HSP70 and HAP1 did not require the
addition of ATP. The association of HSP70 and a key base excision
repair enzyme suggests a role for heat shock proteins in
promoting base excision repair. These findings provide a possible
mechanism by which HSP70 protects cells against oxidative stress.
HSP701 is a member of a
family of proteins, the transcription of which is stimulated in
response to heat shock, oxidation, and other stresses in eukaryotes and
prokaryotes (1, 2). Upon association, HSPs alter the folding of some
proteins and often enhance their biological functions (3). In general,
the chaperone functions of HSPs improve cellular responses to stress. HSP70 is an ATPase and plays a role in the transport of certain proteins within the cell (4). Although HSP70 helps protect cells
against oxidative stress, it is unclear how it functions in this role
(3).
Base excision repair (BER) is a major repair pathway and is responsible
for correcting much of the DNA damage caused by ionizing radiation and
reactive oxygen species. Much of the core enzyme machinery involved in
BER has been described; however, the regulation and coordination of BER
with other cellular processes is not well understood. HAP1 (also known
as Ape1 and Ref-1) is the first enzyme in the BER pathway that is
utilized to repair most if not all of the types of damage acted on by
this process. Thus, it is a good candidate for a target of regulation.
We previously reported that human HSP70 could bind to HAP1 as
determined by affinity chromatography and hydroxyl radical footprinting (5). Here we confirm the association using immunoprecipitation and
electrophoretic mobility shift assays. HSP70 also markedly enhanced the
specific endonuclease activity of HAP1. This report and our previously
reported results (5) are the first indication of a role for HSPs in BER
in human cells.
Materials--
Recombinant human HAP1 (His6-tagged)
was expressed in Escherichia coli and purified to apparent
homogeneity. The HAP1 expression plasmid was a kind gift from Dr. Ian
Hickson (University of Oxford, United Kingdom). Purified human
uracil-DNA glycosylase, UDG AP Endonuclease Assay--
HAP1 AP endonuclease activity was
assayed by the conversion of supercoiled, depurinated pBR322 to a
nicked form. Depurination of the DNA to achieve ~1
depurination/plasmid (i.e. 1 event/4363 bp) was done in 10 mM sodium citrate (pH 5.0) and 100 mM NaCl at
70 °C for 10 min. Plasmid DNA that acquired
Supercoiled, depurinated pBR322 DNA (100 ng) in a 20-µl reaction
volume was digested with HAP1 at the indicated concentrations for 30 min at 37 °C in 66 mM Tris (pH 7.5), 5 mM
MgCl2, and 1 mM Immunoprecipitation and Western Blot--
Highly purified
recombinant HSP70 (0.5 µg) and highly purified recombinant HAP1 (0.5 µg) were incubated in phosphate-buffered saline at 4 °C for
16 h. They were then coprecipitated on protein A-Sepharose beads
(Amersham Pharmacia Biotech) using antibodies against HSP70 or HAP1 as
indicated. The Sepharose beads were washed four times using
phosphate-buffered saline containing 0.1% Nonidet P-40 with brief
centrifugation at 15,500 × g. The firmly bound proteins were eluted from the beads by boiling for 5 min in gel electrophoresis loading buffer containing 2% SDS, 630 mM
Samples were electrophoresed on a 7.5% polyacrylamide-SDS gel for
~2.5 h with molecular weight markers (Bio-Rad). The separated proteins were then transferred electrophoretically to nitrocellulose membranes. The nitrocellulose membranes were sequentially blocked with
5% milk proteins, incubated with rabbit anti-HSP70 antibody, rinsed,
and incubated with the secondary antibody (donkey anti-rabbit IgG
precoupled to horseradish peroxidase), which was detected by
chemiluminescence (ECL, Amersham Pharmacia Biotech) using x-ray film
(Kodak XAR5). As a specificity control, bovine serum albumin (0.5 µg)
was substituted for the protein that was omitted from the
immunoprecipitation reaction. For Western blot immunostaining, anti-HSP70 (rabbit antiserum) was used at a 1/20,000 dilution, and the
secondary antibody (donkey anti-rabbit) was used at a 1/10,000 dilution.
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assays were performed using HAP1, HSP70, and a
double-stranded 30-bp oligonucleotide containing a single uracil 12 nucleotides from the 5' terminus of the top strand of sequence 1.
The U residue was converted to an AP site by preincubation of the 30-bp
32P-labeled oligonucleotide (1.5 pmol) with UDG (3.0 pmol)
in reactions (20 µl) containing 50 mM Hepes-KOH (pH 7.8),
1 mM EDTA, and 5 mM dithiothreitol for 5 min at
37 °C. HAP1 (3.3 pmol) was then added to reaction mixtures and
incubated for 30 min at 0 °C. Note that the low temperature and the
presence of EDTA prevented the cleavage of AP sites by HAP1. HSP70 was
then added as indicated, and incubations were continued for an
additional 30 min at 0 °C. Loading dye (5× = 0.25% xylene cyanol,
0.25% bromphenol blue, 10 mM EDTA, and 50% glycerol) was
added, and mixtures were electrophoresed on 5% nondenaturing
acrylamide gels at 30 mA for 1.5 h. The electrophoresis buffer
consisted of 6.7 mM Tris (pH 6.8), 3.3 mM
sodium acetate, and 1 mM EDTA. 32P-Labeled
bands in the gel were quantitated by densitometer scans (Molecular
Dynamics ImageQuant) of autoradiograms.
Coimmunoprecipitation of HSP70 and HAP1--
The association of
HSP70 and HAP1 was investigated by immunoprecipitation/Western blotting
(Fig. 1). Purified HSP70 and HAP1 were
mixed and immunoprecipitated using protein A-Sepharose beads and
antibodies against either HSP70 or HAP1. The immunoprecipitates were
examined by SDS-polyacrylamide gel electrophoresis and immunoblotting using antiserum against HSP70. An interaction between HSP70 and HAP1
was demonstrated by the coprecipitation of the two proteins by
anti-HAP1 antibody (Fig. 1, lane 6). The positive signal was dependent on both HSP70 and HAP1 ( Fig. 1, compare lanes 4 and 5 with lane 6). The HSP70 was not merely
acting "sticky" because it did not appreciably bind to and
precipitate with the protein A-Sepharose and anti-HAP1 antibody when
HAP1 was not present (Fig. 1, lane 5). Anti-HSP70
precipitated HSP70 when present (Fig. 1, lanes 2 and
3) as expected. Surprisingly, however, more HSP70 was pulled
down in the presence of HAP1 than in its absence. It is possible that
the binding of HAP1 to HSP70 alters the conformation of the latter
protein to make it more accessible to the antibody. These results are
consistent with our previous findings of an HSP70-HAP1 association as
detected by affinity chromatography and hydroxyl ion footprinting
(5).
HSP70- and HAP1-dependent Complex Formation with
AP-DNA--
To further investigate the interaction between HSP70 and
HAP1 and the functional consequences of this interaction, we employed an electrophoretic mobility shift assay using a duplex oligonucleotide containing an AP site. A 30-bp 32P-labeled oligonucleotide
was first pretreated with UDG to remove a single uracil moiety and
create an AP site at position 12 of the labeled strand. HAP1 and/or
HSP70 were incubated with the AP oligonucleotide in the presence of
EDTA to prevent AP endonuclease action. Fig.
2A shows that a clear mobility
shift occurs in the presence of both HAP1 and HSP70. This mobility
shift is also dependent on an AP site as demonstrated by the
requirement for pretreatment of the DNA with UDG. Likewise, a mobility
shift was not observed when the AP oligonucleotide was used in a
single-stranded state (data not shown). A long exposure (180 versus 1.5 min) of the gel revealed that a small amount of
protein-DNA complex formed in the presence of HAP1 and AP
oligonucleotide without any HSP70 (Fig. 2B, lane
3). The fact that this complex exhibited the same mobility in the
presence or absence of HSP70 suggests that HSP70 promotes the formation
of a HAP1-DNA complex but does not remain part of that complex.
Consistent with this interpretation, immunoblot studies indicated the
presence of HAP1 and the absence of HSP70 in the shifted complex (data
not shown). We also found that incubation of an AP oligonucleotide with
very high levels of HAP1 led to formation of a substantial amount of
HAP1-DNA complex. Other investigators have previously observed
retardation of oligonucleotides with AP sites when incubated with HAP1
alone (6-8). The data presented here demonstrate that HSP70 markedly
increases the amount of HAP1-DNA complex formed. We found that HSP70
itself exhibited no detectable affinity for DNA containing AP sites or
nicks in mobility shift assays.
Fig. 3 shows that incubation of the AP
oligonucleotide with HAP1 and increasing amounts of HSP70 enhanced the
fraction of the DNA that formed a complex with HAP1. Incubation at
37 °C augmented HAP1-DNA complex formation compared with incubation
at 0 °C. Overall, HSP70 stimulated formation of a HAP1-DNA complex
by at least 50-fold.
Fig. 4 demonstrates that the amount of
HAP1-DNA complex formed is directly proportional to the quantity of
HAP1 present in the mobility shift assay. Because HSP70 is a known
ATPase, the effect of ATP on the assay was also examined. ATP did not
affect the HSP70-dependent formation of HAP1-DNA complex
(Fig. 4).
Stimulation of HAP1 Endonuclease Activity by HSP70--
We found
that an important consequence of the interaction between HSP70 and HAP1
was enhanced endonuclease activity of HAP1 at AP sites (Fig.
5). The substrate for HAP1 was plasmid
DNA containing abasic sites (~1 AP site/plasmid). HAP1 nicking at AP
sites was detected by the conversion of covalently closed circular DNA
to a more slowly migrating, nicked circular form in agarose gels containing ethidium bromide. HSP70 markedly stimulated HAP1 activity, whereas HSP70 by itself exhibited no AP endonuclease activity (Fig.
5A). Quantitation of these results indicates that HSP70 caused a 10-100-fold enhancement of HAP1 activity (Fig.
5B). Bovine serum albumin did not alter the nicking ability
of HAP1 (data not shown). In Fig. 6, HAP1
was titrated at various levels of HSP70. The stimulation of HAP1
endonuclease by HSP70 was dose-dependent. HSP70 also
increased HAP1 nicking of a duplex oligonucleotide containing an AP
site (data not shown).
The 70-kDa heat shock proteins are molecular chaperones that are
involved in many aspects of protein activity (9). The physical
association of HSP70 with HAP1 as well as the enhancement of HSP70 for
HAP1 endonuclease activity at abasic sites demonstrated here are novel
findings that suggest a role for HSP70 in the BER of DNA in human
cells. The enhancement of HAP1 and BER by HSP70 may help explain the
protective role of HSP70 against oxidative stress (3).
Some of the HSP70s possess weak ATPase activity (9), but HSP70 alone
exhibited no endonuclease activity under the conditions of these
experiments, and ATP was not required in the studies presented here.
HSP27 also stimulated HAP1 endonuclease activity in experiments similar
to those with HSP70 (data not shown). This might be related to common
structural features in the HSPs. HSP70 did not stimulate the
endonuclease activity of the E. coli AP endonuclease IV
protein, indicating a specificity in the interaction between human
HSP70 and HAP1.
A single-stranded DNA-binding protein was required for human DNA
excision repair (10), and molecular chaperonins have been implicated in
nucleotide excision repair in E. coli (11, 12). Therefore, a
role for accessory proteins in BER was not entirely unexpected. From
data provided here, HSP70 appears to be an accessory protein involved
in BER in human cells.
The mechanisms by which HSP70 enhanced attachment of HAP1 to AP sites
in DNA and enhanced its enzymatic attack are not understood. Furthermore, the steps by which HAP1 itself interacts with AP sites in
DNA are complex. Recent results from Demple and co-workers (7, 8) have
greatly clarified these steps by demonstrating that HAP1 has a high
affinity for incised abasic sites and that the incised DNA product acts
as a competitive inhibitor for HAP1. Relief of the inhibition by DNA
polymerase The interaction of HSP70 and HAP1 could have broad implications for
understanding how BER works in human cells. The enhancement of DNA
repair in E. coli by induction of DnaK/J synthesis
(12) and the heat shock enhancement of DNA repair and resistance to oxidative damage in pancreatic islet cells (3, 13) may be related to
the specific DNA repair activity of HAP1 described in this report.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
84, a recombinant enzyme lacking 84 amino
acids at the N terminus, was a generous gift from Dr. G. Slupphaug
(UNIGEN, University of Trondheim, Norway). Recombinant human HSP70 and
rabbit antiserum against HSP70 were purchased from StressGen (Victoria,
British Columbia, Canada). Rabbit IgG against HAP1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Bovine serum albumin was obtained from
Sigma. USB T4 polynucleotide kinase and [
-32P] ATP
(3000 Ci/mmol) were from Amersham Pharmacia Biotech. DNA oligonucleotides were prepared at the Albert Einstein DNA Sequencing and Oligonucleotide Facility. Plasmid pBR322 was from Roche Molecular Biochemicals.
1 abasic site/plasmid by this treatment was converted to a nicked form by digestion with
HAP1. Approximately 37% (1/e) of the plasmid DNA lacked an AP
site and remained resistant to HAP1 treatment as expected when an
average of 1 abasic site/plasmid was present.
-mercaptoethanol. Reactions
were stopped by addition of 5 µl of neutral loading buffer (0.25%
xylene cyanol, 0.25% bromphenol blue, 10 mM EDTA, and 50%
glycerol). Samples were then heated for 5 min at 60 °C and
electrophoresed for 3 h at 90 V on a 0.8% agarose gel containing
ethidium bromide. The covalently closed and nicked forms were
visualized by UV light and photographed. The photographic negative was
scanned using a Molecular Dynamics ImageQuant densitometer.
Densitometry values were obtained, and the ratios of nicked DNA to the
total DNA were calculated.
-mercaptoethanol, 10% glycerol, 62.5 mM Tris (pH 6.8),
and 0.0025% bromphenol blue.
The top strand was 5'-32P-end labeled using T4
polynucleotide kinase and standard procedures. Unincorporated
radiolabel was eliminated by passage of the labeled oligonucleotide
through Sephadex G-50. The labeled oligonucleotide was annealed with
the unlabeled complementary oligonucleotide containing a G residue
opposite the U.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Coimmunoprecipitation of HAP1 and HSP70.
Immunoprecipitation/Western blotting was done as described under
"Experimental Procedures." HAP1 and/or HSP70 were coincubated and
immunoprecipitated using antibodies to one protein or the other as
indicated. Bovine serum albumin (BSA) was substituted for
the omitted protein. The immunoprecipitates were subjected to
SDS-polyacrylamide gel electrophoresis. Immunoblots were carried out
using antibodies against HSP70. (Note that in addition to detecting
HSP70, the secondary antibody (donkey anti-rabbit IgG) also revealed
the IgG (rabbit) heavy chains from the immunoprecipitates.)
Quantitative densitometer scans of the HSP70 bands demonstrated the
relative amount of HSP70 that was precipitated: lane 1, 0 units; lane 2, 83 units; lane 3, 350 units;
lane 4, 0 units; lane 5, 6 units; lane
6, 335 units.
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Fig. 2.
Altered electrophoretic mobility of an AP
oligonucleotide in the presence of HAP1 and HSP70. The effect of
HAP1 and HSP70 on the electrophoretic mobility of an AP oligonucleotide
was tested. A 30-bp 5'-32P-labeled oligonucleotide
containing a uracil residue at position 12 of the labeled strand was
pretreated with UDG (lanes 2-5) to create an AP
site. HAP1 (lanes 3, 5, and 6, 3.3 pmol) and/or HSP70 (lanes 4-6, 29 pmol) were added to
reactions and incubated at 0 °C in the presence of 10 mM
EDTA as described under "Experimental Procedures." Reaction
mixtures were electrophoresed on a 5% native polyacrylamide gel, and
an autoradiogram is shown. A, 1.5-min exposure.
B, 180-min exposure. Note the 30-bp "free" DNA and the
asterisk indicating the protein-DNA complex.
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Fig. 3.
Dependence on HSP70 for HAP1-DNA complex
formation. A, a titration of HSP70 in the
electrophoretic mobility shift assay was performed as described in Fig.
2 and under "Experimental Procedures." The 32P-labeled,
uracil-containing oligonucleotide was pretreated with UDG to create
abasic sites. Reactions were adjusted to 5 mM EDTA, HAP1
was added, and incubations were continued at 0 °C for 30 min.
Reaction mixtures then received increasing amounts of HSP70 (as
indicated in B) and were incubated for another 30 min at
37 °C (lanes 1-5) or 0 °C (lanes 6-10)
followed by electrophoresis of the samples and autoradiography. Note
the 30-bp "free" DNA and the asterisk indicating the
protein-DNA complex. B, quantitation of results from
A: incubation at 37 °C ( , lanes 1-5) and
incubation at 0 °C (
, lanes 6-10).
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Fig. 4.
Effect of HAP1 concentration and ATP on the
electrophoretic mobility shift assay. A titration of HAP1 in the
presence of HSP70, with and without ATP, was performed as described in
Fig. 2 and under "Experimental Procedures." The
32P-labeled, uracil-containing oligonucleotide was
pretreated with UDG to create abasic sites. Reactions were adjusted to
10 mM EDTA. HAP1, ATP (2 mM), and HSP70 (14 pmol) were added as indicated, and mixtures were incubated at 0 °C
for 40 min followed by electrophoresis of the samples and
autoradiography.
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Fig. 5.
Effect of HSP70 on HAP1 cleavage of AP sites
in plasmid DNA. A, covalently closed plasmid DNA
containing ~1 AP site/plasmid was preincubated at 0 °C for 60 min
with or without HSP70 (1 µg) and serial dilutions of HAP1
(lanes 1 and 7, 3.3 pmol; lanes 2 and
8, 0.33 pmol; lanes 3 and 9, 33 fmol;
lanes 4 and 10, 3.3 fmol; lanes 5 and
11, 0.33 fmol; lanes 6 and 12, 0.033 fmol; lanes 13-16, no HAP1). The mixture was incubated for
30 min at 37 °C before electrophoresis on an agarose gel containing
ethidium bromide. Note the nicked circular and the covalently closed
circular (CCC) DNAs. B, quantitation
of the results in A. , +HSP70;
,
HSP70.
(+HSP70)
and
(
HSP70) represent values obtained in a repeat experiment. The
20 µl of reaction mixture with HAP1 at 10
2 dilution
contained 3.3 pmol of HAP1.
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Fig. 6.
Stimulation of HAP1 endonuclease activity at
various levels of HSP70. The AP endonuclease assay was performed
as described in Fig. 5 and under "Experimental Procedures." Serial
dilutions of HAP1 were carried out in the presence of different amounts
of HSP70. The reaction with HAP1 at a 10 2 dilution
contained 3.3 pmol of HAP1. The amounts of HSP70 used were:
, 0.5 µg;
, 0.2 µg;
, 0.04 µg; and
, none.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was demonstrated. HSP70 might also relieve product
inhibition. Alternatively, HSP70 may alter the configuration of HAP1 to
a more active one. The continued binding of HSP70 to HAP1 seems not to
be required once the correct enzyme placement occurs at the AP site.
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FOOTNOTES |
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* This work was supported in part by Core Support Cancer Research Center Grant P30 CA 13330 from the National Cancer Institute and by the Rome Sisters Foundation for Cancer Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Grant CA 71612.
Supported by National Institutes of Health Grant CA 52025.
** To whom correspondence should be addressed: Montefiore Medical Center, Albert Einstein College of Medicine, 111 E. 210 St. Bronx, New York 10467. Tel.: 718-920-2641; Fax: 718-655-4261; E-mail: franklin@aecom.yu.edu.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M009297200
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ABBREVIATIONS |
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The abbreviations used are: HSP70, human heat shock protein 70; BER, base excision repair; HAP1, human apurinic/apyrimidinic endonuclease 1; AP, apurinic/apyrimidinic; bp, base pair(s); UDG, uracil-DNA glycosylase.
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REFERENCES |
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