apyrimidinic endonuclease by heat shock protein 70

DNA Repair 2 (2003) 259–271

Specific stimulation of human apurinic/apyrimidinic endonuclease by heat shock protein 70 Frances Mendez a , Margarita Sandigursky a,b , Raichal P. Kureekattil c , Mark K. Kenny c , William A. Franklin a,b , Robert Bases a,b,∗ a

c

Department of Radiation Oncology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210 Street, Bronx, NY 10467, USA b Department of Radiology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210 Street, Bronx, NY 10467, USA Department of Emergency Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210 Street, Bronx, NY 10467, USA Accepted 28 October 2002

Abstract We previously demonstrated the stimulation of human apurinic/apyrimidinic endonuclease 1 (HAP1) by heat shock protein 70 (HSP70). In this work, we further defined the functional interaction between these proteins. Digestion of HSP70 by trypsin released 48 and 43 kDa amino terminal fragments that retained the ability to stimulate HAP1. In agreement with this result, an HSP70 N-terminal deletion mutant protein containing amino acids 1–385 was comparable to the full-length protein in its ability to enhance HAP1 activity. HSP70 mutants containing carboxy terminal amino acids 386–640 stimulated HAP1 only slightly, as did unrelated proteins. These results implicate the amino terminal portion of HSP70 in stimulating the activity of HAP1. © 2002 Elsevier Science B.V. All rights reserved. Keywords: HAP1 protein; HSP70 protein; Heat shock protein; AP site; Base excision repair

1. Introduction HAP1, also described as APE1/REF-1, is responsible for a crucial step in DNA base excision repair by performing single-stranded scission at the abasic sites that remain after glycosylase excision of damaged bases. Succeeding steps include removal of

Abbreviations: HAP1, human apurinic/apyrimidinic endonuclease 1; HSP70, human heat shock protein 70; BER, base excision repair; AP, apurinic/apyrimidinic; bp, base pair(s) ∗ Corresponding author. Tel.: +1-718-920-2641; fax: +1-718-655-4261. E-mail address: [email protected] (R. Bases).

5 -phosphosugar remnants and gap filling by DNA polymerase ␤, followed by ligation of the replacement nucleotide(s). HSP70 binds to HAP1 and greatly stimulates its endonuclease activity [1,2]. HSP70 is a highly conserved cellular protein whose synthesis is enhanced by stress conditions (e.g. heat shock, ionizing radiation, oxidative insults, heavy metal exposure), many of which damage DNA. The ability of HSP70 to intervene in the activities of many biologically important protein molecules is ascribed to its chaperone functions, by which folding or unfolding of client molecules is achieved [3–5]. By stimulating HAP1, HSP70 may be an important aid to the repair of DNA damage in cells. Although the

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chaperone activity of HSP70 generally depends on its intrinsic ATPase activity, ATP was not necessary for stimulation of HAP1 activity [1]. Stimulation of other proteins by HSPs has been documented to be ATP-independent [6,7]. Beyond enhancing the binding of HAP1 to damaged DNA, the mechanism by which HSP70 stimulates the HAP1 AP endonuclease activity is not known. To address this question, we wished to determine which portion of the HSP70 molecule is required for HAP1 stimulation. Three functional domains have been identified within HSP70 [7–9]. The amino terminal half (∼44 kDa) of the protein harbors the ATPase activity. The substrate binding region (∼15 kDa) is towards the carboxy terminus. The extreme COOH-terminal end is a regulatory domain. This region is highlighted by the terminal four amino acids, EEVD, which are completely conserved in all eukaryotic HSP70 molecules. The EEVD regulatory region controls ATPase activity, substrate binding, HSP70 conformation, and interaction with cochaperone proteins (e.g. HSP40). The three-dimensional structure of the protein has been compared to a pot (the ATPase domain) with a lid (the remaining C-terminal domains). Here we report that the N-terminal pot-shaped domain of HSP70 is more important for stimulation of HAP1 than the C-terminal lid-shaped portion. 2. Materials and methods 2.1. Enzymes and reagents Recombinant human HAP1 (His6 -tagged) was expressed in E. coli and purified to apparent homogeneity [1,2]. The HAP1 expression plasmid was a kind gift from Dr. Ian Hickson (University of Oxford, UK). Recombinant human HSP70 was purchased from StressGen (Victoria, BC, Canada). N-terminal (amino acids 1–385) and C-terminal (amino acids 386–640) mutant HSP70 proteins [7,8] were a generous gift from Dr. Jaewhan Song and Dr. Richard I. Morimoto, Northwestern University. Prior to use, these proteins were pre-incubated at 50 ◦ C for 10 min to eliminate any nuclease activities. Plasmid pBR322 (4363 bp) was from Roche Molecular Biochemicals. Bovine pancreatic TPCK trypsin immobilized on 4% cross-linked beaded agarose was purchased from

Pierce. Protein molecular weight standards were purchased from Bio-Rad. Phagemid pBKS was from Stratagene. 2.2. Digestion of HSP70 by immobilized trypsin Immobilized trypsin slurry (50 ␮l, 1.25 mg trypsin protein, 300 units) was washed three times with 500 ␮l of 50 mM sodium phosphate, pH 7.0. HSP70 was incubated in the same buffer with the washed immobilized trypsin and digested at 37 ◦ C for 18 h. The trypsin was eliminated by centrifugation and the digested HSP70 fragments were harvested in the supernatant. Aliquots of the proteolytic digests were resolved on 12% polyacrylamide/SDS gels, and were subsequently silver stained. 2.3. Plasmid AP endonuclease assay HAP1 AP endonuclease activity was assayed by the conversion of the covalently closed circular (CCC) depurinated pBR322 or pBKS to a nicked circular form. Depurination of the DNA to achieve approximately one depurination per plasmid pBR322 (i.e. one event/4363 bp) was performed as described previously [1]. Plasmid DNA that acquired one or more abasic sites per plasmid by this treatment was converted to a nicked circular 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 one abasic site per plasmid was present. Supercoiled depurinated pBR322 DNA (100 ng) in a 20 ␮l reaction volume was digested with HAP1 at indicated concentrations for 30 min at 37 ◦ C in 66 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , and 1 mM ␤-mercaptoethanol. In some experiments HAP1 and HSPs were first bound to depurinated pBR322 for 10 min on ice, followed by incubation at 37 ◦ C for 3 min. Reactions were stopped by addition of 5 ␮l of neutral loading buffer (0.25% xylene cyanol, 0.25% bromophenol blue, 10 mM EDTA, and 50% glycerol). Samples were then heated for 5 min at 60 ◦ C and resolved 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

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values were obtained, and the ratios of nicked DNA to the total DNA were calculated.

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[1]. Formamide was added to reaction mixtures which were boiled for 5 min, quenched on ice and resolved on 20% acrylamide gels containing 7 M urea.

2.4. Glycerol gradient sedimentation Digests of HSP70 (200 ␮l) were placed on a 5 ml of 10–30% neutral glycerol gradient containing 50 mM sodium phosphate, pH 7.0, 0.1 mM Na2 EDTA and sedimented for 21 h at 45,000 rpm at 4 ◦ C in a Beckman SW50.1 rotor. Fractions of 0.25 ml were collected from the bottom of the gradient. Protein standards (97, 66, 45, 31, 21.5 and 14.4 kDa) were resolved in a gradient under identical conditions. 2.5. N-terminal amino acid determination Edman degradation and identification of the eight N-terminal amino acids of HSP70 and its 48 and 43 kDa digestion products were determined by standard methods. These reactions were performed at the Laboratory of Macromolecular Analysis and Proteomics at the Albert Einstein College of Medicine with the kind assistance of Dr. George Orr and Linda Siconolfi-Baez. 2.6. Electrophoretic mobility shift assay Electrophoretic mobility shift assays were performed using HAP1, HSP70, and a double-stranded 30 bp oligonucleotide containing a single uracil at position 12 (given in bold) as shown: 5 -ATATACCGCGGUCGGCCGATCAAGCTTATT-3 3 -TATATGGCGCCGGCCGGCTAGTTCGAATAA-5

The uracil-containing strand was 5 -32 P-end-labeled using T4 polynucleotide kinase and standard procedures. Unincorporated radio-labeled nucleotide was eliminated by passage of the labeled oligonucleotide through Sephadex G-50. The labeled oligonucleotide was annealed with the unlabeled complementary oligonucleotide containing a guanine residue opposite the uracil. Uracil was removed to produce an AP site by pre-incubation of the 30 bp double-stranded oligonucleotide (1.5 pmol) with UDG (3.0 pmol) in a reaction (20 ␮l) containing 50 mM HEPES–KOH, pH 7.8, 1 mM Na2 EDTA, 5 mM DTT for 5 min at 37 ◦ C. HAP1 endonuclease digestions were then performed

3. Results 3.1. Stimulation of HAP1 by N-terminal tryptic fragments of HSP70 HSP70 was digested with immobilized trypsin (Fig. 1A) and the digest was then tested for stimulation of HAP1 endonuclease activity. Serial dilutions of HAP1 were tested for the ability to cleave pBR322 DNA plasmid containing AP sites to relaxed forms with or without HSP70 or trypsin-digested HSP70 (Fig. 1B). Recovery of 43 and 48 kDa trypsin fragments was determined by comparing their band densities with the HSP70 standards in lanes 1–4 of Fig. 1A. The 43 and 48 kDa digestion fragments and the residual trypsin-resistant HSP70 are shown in lanes 5–7. Together these three species account for 39% of the HSP70-derived proteins placed on the gel. Edman analysis of the 48 and 43 kDa tryptic digestion products showed that their eight N-terminal amino acids were identical to those of full-length HSP70. With the Edman technique serial stepwise chemical removal of N-terminal amino acids is achieved. The amino acids are identified and compared with standards. The N-terminal amino acid sequences of both the 43 and 48 kDa fragments were identical to that of native human HSP70. The chain starts at residue 2 (minus the initiating methionine) as: AKAAAVGI. These results suggested that it was the N-terminal portion of HSP70 which was active in stimulating HAP1. Loss of approximately 36% of the gel input could be accounted for by the elimination by trypsin of the 25 kDa C-terminal portion of HSP70. The trypsin-resistant HSP70 fraction represents 19% of the proteins found in gel lanes 5–7. No smaller proteins could be detected in these lanes. The C-terminal portion of HSP70, 25 kDa, was not found; presumably it had been digested to small peptides and therefore could not contribute to the HAP1 stimulation shown in Fig. 1B. Despite enzymatic truncation, the shortened HSPs exhibited little or no loss of their ability to enhance HAP1/plasmid nicking activity. Stimulation

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Fig. 2. HSP70 dose-dependent enhancement of HAP1 cleavage at AP sites. Three separate AP endonuclease assays were done to show HSP70 enhanced relaxation of covalently closed plasmid containing approximately one AP site per plasmid exposed to 10−5 dilution of HAP1 (20 fmol/20 ␮l reaction volume) as described in Section 2.3 (•, 䊉, 䊊). Plasmid, HAP1 and the indicated amounts of HSP70 were incubated together on ice for 10 min and then at 37 ◦ C for 3 min before stopping the reaction with loading buffer. No HSP70 and no HAP1 (䉲); HAP1 at the rate of 10−5 (20 fmol), and no HSP (䊏). Error terms are for the mean and standard error of the mean values (0.5 ␮g of HSP70 is 7.0 pmol).

of HAP1 by small peptides <10 kDa, derived from digestion, is excluded by results shown later in Fig. 3. The fraction of nicked plasmids shown in Fig. 1B did not exceed 80%, despite the presence of the high concentrations of HAP1 in the least diluted test aliquots (i.e. 10−2 and 10−3 dilutions). The 20% of plasmids which were not nicked reflects the plasmid fraction which resisted HAP1 digestion because it had previously escaped depurination and depyrimidination during heat and low pH treatment. Heat and low pH treatment conditions had been chosen to spare 0.37 of the plasmids [1]. Approximately 25% of plasmid pBR322 molecules had been nicked before HAP1 di-

gestion, constituting the background value due to hydrolysis during preparation and handling. Therefore, the plateau ceiling of 80% nicked plasmids shown in Fig. 1B reflects a net increase of 0.80 − 0.25 = 0.55 of nicked plasmids over background values, in good agreement with the 0.63 expected when one depurination/depyrimidination per plasmid is achieved. Stimulation of HAP1 was directly dependent upon HSP70 concentration. Results of the experiments of Fig. 2 established the dependence of HAP1 plasmid nicking upon HSP70 concentration. To confirm that the 48 and 43 kDa trypsin products stimulated HAP1 cleavage of AP site-containing

䉳 Fig. 1. Stimulation of HAP1 endonuclease activity by HSP70 tryptic fragments. (A) Release of 43 and 48 kDa tryptic digest fragments from HSP70. HSP70 (40 ␮g) in 150 ␮l of 50 mM sodium phosphate, pH 7.0, including 50 ␮l of washed immobilized TPCK trypsin, was incubated for 18 h at 37 ◦ C. After centrifugation to pellet the immobilized trypsin, the supernatant containing the digestion products was subjected to another digestion. Aliquots of the digestion products were resolved by 12% SDS-PAGE and the gel was stained with silver nitrate. Lanes 1–4 contain 500, 250, 125 and 63 ng of undigested HSP70, respectively. The estimated gel input of HSP70 to lanes 5–7, prior to digestion by trypsin, was 1200, 800 and 400 ng, respectively. Lanes 5–7 were found to contain 470, 313 and 157 ng HSP70 trypsin digestion products. Lane M, generic protein molecular weight markers. (B) Stimulation of HAP1 endonuclease activity with intact HSP70 and tryptic fragments. The AP endonuclease assay was performed with AP site-containing pBR322 as described in Section 2.3. Serial dilutions of HAP1 were carried out in the presence of 0.5 ␮g of either intact HSP70 or trypsin-digested HSP70. HAP1 digestions were supplemented with intact HSP70 (•), digested HSP70 (䉱), no HSP70 (䊊), and supernatant from trypsin beads which had been incubated without HSP70 for 18 h at 37 ◦ C (䉭). A 20 ␮l reaction mixture with HAP1 at 10−2 dilution contained 20 pmol of HAP1.

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Fig. 3. Glycerol gradient separation of trypsin-digested HSP70 products. HSP70 (40 ␮g) in 250 ␮l of 50 mM sodium phosphate, pH 7.0, was digested for 18 h at 37 ◦ C. The supernatant was sedimented for 18 h on a 5 ml of 10–30% glycerol gradient in a swinging bucket SW50.1 rotor at 45,000 rpm at 4 ◦ C. A total of 20 fractions (0.25 ml each) was collected from the bottom of the gradient. (A) Gradients fractions (25 ␮l) were resolved on a 12% SDS-PAGE gel. Lanes 17–19 contain 0.06, 0.125 and 0.25 ␮g undigested HSP70, respectively. Lane M, molecular weight markers. (B) Stimulation of HAP1 endonuclease activity with HSP70 tryptic fragments. Aliquots of the gradient fractions (10 ␮l) were first bound to HAP1 (20 fmol/20 ␮l reaction volume) and subsequently incubated at 37 ◦ C with AP site-containing pBR322 DNA. Gradient fractions were also tested in the absence of HAP1 (lower panel). Nicked circular and covalently closed circular (CCC) DNAs are indicated. (C) Quantitation of the results in (B) in the presence (•) or absence (䊊) of HAP1. The lower panel shows the corresponding protein concentration of the three HSP70 species in individual gradient fractions: HSP70 (䉲); 48 kDa fragment (䉱); 43 kDa fragment (䊏).

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Fig. 3. (Continued ).

DNA, HSP70 was first digested with immobilized trypsin and the digestion products were then separated by velocity sedimentation. As seen in Fig. 3A, the 48 and 43 kDa HSPs sedimented more slowly on the glycerol gradient than the full-length HSP70, which is consistent with release of the C-terminal tryptic residues. The proteins released from HSP70 by trypsin and distributed in the gradient fractions were assayed for their ability to stimulate HAP1 endonuclease activity on AP site-containing plasmids (Fig. 3B and the upper panel of Fig. 3C). The enhancement of HAP1 activity by gradient fraction 13 is due to 40 ng of intact HSP70, with the possibility of only a minor contribution from the small amount of 48 and 43 kDa fragments present. By contrast, the stimulatory activity in fraction 17 must be due to the new 48 and 43 kDa species since it could not be accounted for by trace amounts of HSP70 present and the HAP1 stimulation dependence on HSP70 concentration shown in Fig. 2. 3.2. Enhanced nicking by HAP1 and HSP70 HSP70 stimulation of HAP1 nicking of AP sites in pBR322 was found in time course experiments (Fig. 4A and B). Equimolar amounts of BSA did not

stimulate HAP1 in these studies but BSA did weakly stimulate HAP1 when the HAP1 concentration was 10-fold greater (Fig. 4C). 3.3. Stimulation of HAP1 by the N-terminal mutant of HSP70 To confirm that the N-terminal pot portion of the HSP70 molecule was more important in stimulation of HAP1 activity, we used deletion mutants of HSP70. In addition to the full-length HSP70, we utilized an N-terminal mutant (amino acids 1–385) containing the ATPase domain, and a C-terminal mutant (amino acids 386–640) containing the substrate binding and regulatory domains [7,8]. All proteins were purified after overexpression in E. coli. As shown in the time course in Fig. 5A and B, the N-terminal mutant stimulated HAP1 AP endonuclease activity as well as the intact HSP70 protein. In the absence of HAP1 the N-terminal mutant had no intrinsic AP endonuclease activity. The C-terminal mutant exhibited much weaker stimulatory activity (Fig. 5A). The N-mutant protein of HSP70 achieved three-fold greater stimulation of HAP1 than the C-mutant protein (Fig. 5A). The differences between plateau levels

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Fig. 4. Stimulation of HAP1 activity by HSP70. (A) pBR322 DNA plasmids containing approximately one abasic site each were digested with 20 fmol of HAP1 in 20 ␮l containing HAP1 buffer and 0.25 ␮g of HSP70, or 0.25 ␮g of BSA, or no additions. HAP1 and the protein supplements were incubated together on ice for 1 h before addition of 0.1 ␮g of DNA substrate. Conversion of covalently closed circular plasmid to nicked circular forms at 37 ◦ C proceeded for the times shown. The percentage of nicked forms was determined by electrophoresis in agarose gels, followed by densitometer measurements of ethidium bromide fluorescence (described in Section 2.3). HSP70 + HAP1 (•); BSA+HAP1 (䉱); HAP1 alone (䊏). Untreated plasmid had 29% nicked DNA. (B) An experiment similar to (A), stronger HAP1 stimulation by 0.5 ␮g of HSP70 than with 0.5 ␮g of BSA. Symbols are as in (A). Without HAP1 or other protein additions, 24% of the plasmids exhibited nicked DNA. (C) HAP1 was tested at 200 fmol per reaction. Symbols are as in (A) and (B). When HAP1 was omitted from the reactions, no nicking was obtained with 0.5 ␮g HSP70 (䊊) or with 0.5 ␮g BSA (䉭). With no additions, the substrate had 21% nicked DNA.

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Fig. 4. (Continued ).

shown in the figure were significantly different at P < 0.001, using Student’s t-test, after subtracting the background contribution from HAP1 alone for determinations made with each mutant protein. In the experiment of Fig. 5B, with less HAP1, the N-terminal mutant protein again was significantly more stimulatory than the C-terminal mutant protein. In order of HAP1 stimulation: HSP70 ≥ N-mutant protein ≥ trypsin-derived fragments > C-mutant protein BSA. 3.4. The N-terminal portion of HSP70 stimulates HAP1 AP endonuclease activity on a 30mer oligonucleotide substrate Using an AP site-containing plasmid DNA, we show here that the 385-amino acid mutant protein derived from the N-terminus of HSP70 was more active in stimulating HAP1 than the 255-amino acid C-terminal mutant. The activities of these mutant proteins in stimulating HAP1 activity on a double-stranded oligonucleotide containing a single AP site was measured by detecting digestion fragments in denaturing gels. As seen in Fig. 6, with decreasing levels of HAP1, the N-terminal mutant again showed the most HAP1 stimulatory activity by releasing single-stranded 11mers. Two similar experiments using quantitative densitometry confirmed that the N-mutant protein was more active in stimulating HAP1 than the C-mutant protein or BSA (Fig. 7).

Stimulation of 5 fmol HAP1 by the N-mutant protein in the three experiments with 30mer oligonucleotides shown in Figs. 6 and 7 was 1.24-fold greater than with the C-mutant protein. These results approach significance at P ≤ 0.07 by Student’s t-test. Stimulation of 11mer release by 0.5 and 0.05 fmol in two similar experiments are 1.55- and 1.30-fold, respectively. As would be expected, the N-terminal 43 and 48 kDa proteins derived by trypsin digestion of HSP70 were apparently more active in stimulating HAP1 than the C-mutant protein (Figs. 6 and 7). These results with short linear DNA confirm results obtained with plasmid DNA (Fig. 5). Binding of HAP1 to the 5 -32 P-end-labeled 30 bp oligodeoxynucleotide was dependent upon the abasic site 12 bases from the 5 -end [1]. Those results showed that the binding, as measured by diminution of electrophoretic mobility of resulting HAP1/DNA complex in non-denaturing gels, was greatly stimulated by HSP70. Further experiments, not shown, compared the ability of HSP70, HSP70-derived 48 and 43 kDa trypsin truncated proteins, the C-terminal mutant protein, and BSA to promote HAP1 binding. In accord with the results reported here, the relative stimulatory activity of these proteins on HAP1 binding to the oligonucleotide were: HSP70/trypsin digests/C-mutant proteins/BSA = 1.0/1.45/0.20/0.02. The N-mutant protein could not be tested under these conditions because in the presence of HAP1 it (unexpectedly) substituted for divalent cations; HAP1

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Fig. 5. Effect of HSP70 mutants on HAP1 AP endonuclease activity. A time course of AP endonuclease activity was measured. Each 20 ␮l reaction contained 0.125 ␮g of the indicated protein and 0.1 ␮g of depurinated pBKS DNA. (A) Solid symbols signify addition of 20 fmol of HAP1; open symbols signify no HAP1. Full-length HSP70 (•); N-terminal mutant (䉱, 䉭); C-terminal mutant (䉲); no HSP70, HAP1 alone (䊉); plasmid DNA alone incubated for 30 min at 37 ◦ C (䊐). As a control, plasmid was incubated with the N-terminal mutant protein but no HAP1 (䉭). (B) Symbols are as in (A) except that 2.0 fmol of HAP1 was used.

endonucleolytic attack proceeded despite high levels of EDTA, if the N-mutant protein was present. Alone, the N-mutant protein, HSP70, or the C-mutant protein did not bind to the DNA oligonucleotide and had no detectable endonucleolytic activity.

4. Discussion The HSP70/HAP1 interaction was shown in previous studies [1,2] and confirmed in the present results, in which the N-terminal fragments were released

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Fig. 6. Stimulation of HAP1 activity on a 30mer oligonucleotide substrate. One microgram of each HSP70-related protein was incubated on ice for 20 min in 20 ␮l of buffer containing HAP1 at the dilutions shown. The 5 -32 P-end-labeled 30mer substrate (1 pmol) was added to a reaction mixture containing 66 mM HEPES–KOH, pH 7.5, 5 mM MgCl2 , and 1 mM ␤-mercaptoethanol and was incubated at 37 ◦ C for 30 min. Formamide (15 ␮l) was added to 15 ␮l of the reaction mixture, which was subsequently boiled for 5 min, quenched on ice, and resolved on a 20% polyacrylamide gel containing 7 M urea.

from HSP70 by trypsin. The strategy of using incomplete digestion of proteins by trypsin had also proven useful in determining the structural and functional domains of DNA mismatch repair proteins [10]. Demonstration of the more active pot-like 385-amino acid N-terminal HSP70 mutant protein also clearly implicated this part of the molecule as sufficient for stimulation. The lid-like 255-amino acid C-terminal protein was less active than the N-terminal pot by the determinations described in this report. The mechanism of HAP1 stimulation by HSPs remains unknown. Some aspect of the chaperone function clearly is retained by the pot-like structure. Perhaps it served as a spool for HAP1 to wind around, thereby fully exposing active sites. It is important to learn which structural parts of the HSP70 and HAP1 associate in order to get clues to the separate roles of the lid and the pot structures in the stimulation mechanism of HAP1. For example, loss of the lid structure of DnaK, the 70 kDa molecular chaperone of E. coli, creates an activated form that has a low affinity for

substrate and rapidly binds and releases peptide. Deletion of the lid increased peptide on and off rate constants [11]. Chaperone function was retained by the E. coli chaperone GroEL, even when the client protein 82 kDa aconitase was too large to fit inside the GroEL activating chamber. Stimulation of aconitase activity was observed. Presumably, external features of GroEL were responsible [12], perhaps comparable to results with the lidless HSP70 N-mutant described in this report. The interaction may also be important in determining whether HSP70 provides HAP1 with supplementary nuclear localization signals in addition to those inherent in HAP1 [13]. Enhanced repair of damaged cellular DNA from the HSP70/HAP1 interaction seems probable and could account for the persistent abundance of HSP70 in cells stressed by environmental damage. Stimulation of a DNA base excision repair enzyme by a protein involved in transcription has recently been described [14]. The results reported here implicate the N-terminal pot structure of HSP70 in the stimulation of AP

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Fig. 7. Stimulation of HAP1 by HSP70-related proteins: dose response. Relative amounts of 11mers released by serial dilutions of HAP1 endonuclease, in a repetition of the experiment of Fig. 6 were determined by densitometer measurements. Each HAP1 digestion mixture was supplemented with 1 ␮g of HSP70-related proteins. Tryptic digest (•); HSP70 (䊏); N-mutant (䉱); C-mutant ( ); BSA (䊉). The tryptic digest of HSP70 contained 48 and 43 kDa protein fragments (81%) and undigested HSP70 (19%). The inset at the upper right indicates results obtained with intermediate amounts of HAP1 in a third determination.

endonuclease activity and complex formation by HAP1. These assays clearly showed that potency declined in the order: HSP70 ≥ trypsin-resistant 43– 48 kDa fragments ≥ N-terminal pot-like 385-amino acid protein > C-terminal lid-like protein > BSA. However, these novel results should best be regarded as preliminary until the amino acid sequences involved in the association of HSP-derived proteins and HAP1 are determined. Our results demonstrate that HSP70 stimulated HAP1 10–100-fold. HAP1 is a central enzyme of DNA base excision repair, and is important in protecting the genome from ionizing radiation damage and oxidative damage, perhaps accounting for its widespread conservation in nature of HSP70.

Acknowledgements We thank Dr. Jaewhan Song and Richard I. Morimoto, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, for their generous gift of the N-terminal and C-terminal HSP70 mutant proteins. This work was supported by

the Rome Sisters Foundation for Cancer Research. W.A.F. was supported by National Institutes of Health Grant CA 52025. M.K.K. was supported by National Institutes of Health Grant CA 71612.

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