Heat Shock Protein 70 Binds to Human Apurinic/Apyrimidinic Endonuclease and Stimulates Endonuclease Activity at Abasic Sites*

Mark K. KennyDagger §, Frances MendezDagger , Margarita SandigurskyDagger , Raichal P. KureekattilDagger , Joshua D. GoldmanDagger , William A. FranklinDagger ||, and Robert BasesDagger **

From the Departments of Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, UDGDelta 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 [gamma -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.

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 >= 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.

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 beta -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.

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 beta -mercaptoethanol, 10% glycerol, 62.5 mM Tris (pH 6.8), and 0.0025% bromphenol blue.

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.


<AR><R><C><UP>5′-ATA TAC CGC GG</UP><A><AC><UP><B>U</B></UP></AC><AC>&cjs1142;</AC></A><UP> CGG CCG ATC AAG CTT ATT-3′</UP></C></R><R><C><UP>3′-TAT ATG GCG CCG GCC GGC TAG TTC GAA TAA-5′</UP></C></R></AR>

<UP><SC>Sequence</SC> 1</UP>
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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.


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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.

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.


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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 (black-triangle, lanes 1-5) and incubation at 0 °C (, lanes 6-10).

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).


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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.

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).


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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; black-square, -HSP70. open circle  (+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; black-square, 0.2 µg; black-triangle, 0.04 µg; and open circle , none.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  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.

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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Feige, U., and Polla, B. S. (1994) Experientia (Basel) 50, 979-986
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6. Rothwell, D. G., Hang, B., Gorman, M. A., Freemont, P. S., Singer, B., and Hickson, I. D. (2000) Nucleic Acids Res. 28, 2207-2213[Abstract/Free Full Text]
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8. Masuda, Y., Bennett, R. A. O., and Demple, B. (1998) J. Biol. Chem. 273, 30352-30359[Abstract/Free Full Text]
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12. Chow, K. C., and Tung, W. L. (2000) FEBS Lett. 478, 133-136[CrossRef][Medline] [Order article via Infotrieve]
13. Bellmann, K., Wenz, A., Radons, J., Burkart, V., Kleemann, R., and Kolb, H. (1995) J. Clin. Invest. 95, 2840-2845[Medline] [Order article via Infotrieve]


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