©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ATP Hydrolysis Is Required for the DnaJ-dependent Activation of DnaK Chaperone for Binding to Both Native and Denatured Protein Substrates (*)

(Received for publication, February 10, 1995; and in revised form, June 6, 1995)

Alicja Wawrzynów (1)(§) Bogdan Banecki (1) Daniel Wall (2) (3) Krzysztof Liberek (1) Costa Georgopoulos (3) Maciej Zylicz (1)

From the  (1)Division of Biophysics, Department of Molecular Biology, University of Gdansk, 24 Kladki, Gdansk 80-822, Poland, the (2)Department of Cellular, Viral and Molecular Biology, University of Utah, Medical Center, Salt Lake City, Utah 84132, and the (3)Department de Biochimie Medicale, Centre Medical Universitaire, 1, rue Michel-Servet, 1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Using two independent experimental approaches to monitor protein-protein interactions (enzyme-linked immunosorbent assay and size exclusion high performance liquid chromatography) we describe a general mechanism by which DnaJ modulates the binding of the DnaK chaperone to various native protein substrates, e.g. P, O, , P1, RepA, as well as permanently denatured alpha-carboxymethylated lactalbumin. The presence of DnaJ promotes the DnaK for efficient DnaK-substrate complex formation. ATP hydrolysis is absolutely required for such DnaJ-dependent activation of DnaK for binding to both native and denatured protein substrates. Although ADP can stabilize such an activated DnaK-protein complex, it cannot substitute for ATP in the activation reaction. In the presence of DnaJ and ATP, DnaK possesses the affinity to different substrates which correlates well with the affinity of DnaJ alone for these protein substrates. Only when the affinity of the DnaJ chaperone for its protein substrate is relatively high (e.g. , RepA) can a tertiary complex DnaK-substrate-DnaJ be detected. In the case that DnaJ binds weakly to its substrate (P, alpha-carboxymethylated lactalbumin), DnaJ is only transiently associated with the DnaK-substrate complex, but the DnaK activation reaction still occurs, albeit less efficiently.


INTRODUCTION

Recently it has become clear that the Hsp70 (DnaK homologue) activity is regulated by the DnaJ and GrpE co-chaperones (for review, see Georgopoulos et al.(1994)). Eukaryotic Hsp40 (DnaJ homologue) and Hsp24 (GrpE homologue) have been also identified (Silver and Way, 1993; Bolliger et al., 1994; Lalovaya et al., 1994). In the best studied bacterial system, where all three heat shock proteins were first purified as host factors involved in DNA replication (Zylicz and Georgopoulos, 1984; Zylicz et al., 1985, 1987), DnaK's ATPase activity is stimulated up to 50-fold jointly by the DnaJ and GrpE proteins, whereas DnaJ alone stimulates the DnaK's ATPase activity only up to 2-3-fold (Liberek et al., 1991). Recently, the ability of the eukaryotic Hsp40 protein to stimulate Hsp70s ATPase activity was also shown (Cyr et al., 1992, 1994; Cyr and Douglas, 1994). However, the role of the DnaJ homologues in functioning cooperatively with Hsp70's is still not clear. Several groups have presented evidence that in the presence of ATP, DnaJ inhibits the disassociation of rhodanese, CMLA, (^1)P, and from DnaK (Langer et al., 1992; Hoffmann et al., 1992; Osipiuk et al., 1993; Liberek and Georgopoulos, 1993). Another laboratory, using the same substrate (CMLA) but a different technique, reported that the eukaryotic DnaJ homologue (YdJ1p) and ATP are required for the efficient release of CMLA from Hsp70 (Cyr et al., 1992; Cyr and Douglas, 1994). Since both DnaJ and YdJ1p interact poorly with CMLA it was proposed that if DnaJ-like proteins had a low affinity for a polypeptide substrate, then they would be likely to stimulate the disassociation of Hsp70 from a substrate. However, if a DnaJ-like protein has a high affinity for a substrate it will stabilize a Hsp70-substrate complex (Cyr et al., 1994). Interestingly, DnaJ alone has also been shown to function as a chaperone since it prevents other proteins from aggregation (Langer et al., 1992; Schroder et al., 1993).

Here, we attempt to resolve some of these ambiguities in the chaperone field by presenting a general model for the role of DnaJ in regulating DnaK's substrate binding/release properties. Specifically, we find that DnaJ, in an ATP-dependent reaction, inhibits the dissociation of DnaK from its various protein substrates. This reaction occurs both for native and denatured protein substrates, regardless of whether DnaJ binds with high or low affinity. The ability of DnaJ to stimulate DnaK's substrate binding properties correlates with DnaJ's affinity for the various protein substrates. Specifically, DnaJ will activate DnaK to bind to a substrate for which DnaJ itself has a very low affinity (e.g. CMLA), albeit to a less extent than for substrates to which DnaJ binds more efficiently (e.g. , RepA). During preparation of this article, a paper was published by Szabo et al.(1994) proposing a similar mechanism for DnaK/DnaJ synergistic chaperone action, although their conclusion was based on binding to one denatured substrate, inactivated luciferase. Here, by using both denatured and native protein substrates, we show that the DnaJ-dependent activation of DnaK binding properties is a general phenomenon.


MATERIALS AND METHODS

The DnaK and DnaJ molecular chaperones, as well as all protein substrates were purified as described by Wawrzynow and Zylicz(1995). The modified ELISA and size exclusion HPLC were performed as described by Wawrzynow et al.(1995).


RESULTS

The Influence of DnaJ on DnaK's Substrate Binding Properties

It was previously reported that DnaJ can stabilize the DnaK-substrate complexes in an ATP-dependent manner (Langer et al., 1992; Liberek and Georgopoulos, 1993). To test whether this activity is generally applicable, we tested the binding affinity of a limiting amount of DnaK for various protein substrates in the presence of increasing concentrations of DnaJ (Fig. 1). For these experiments we used three native protein substrates (O, P, and ) and a permanently denatured protein CMLA, which bind to DnaK and DnaJ chaperones with different affinities (see Wawrzynow and Zylicz(1995)). In all cases, in the presence of ATP, DnaJ increased the complex formation between DnaK and any of these protein substrates (Fig. 1). Interestingly, when the binding affinity of DnaK to any of these various protein substrates was tested in the presence of DnaJ and ATP, DnaK's relative binding affinity for each of these four proteins changed and became similar to DnaJ's relative binding affinity for each protein (Wawrzynow and Zylicz(1995), and Fig. 1). For example, in the presence of DnaJ and ATP, DnaK exhibited its highest affinity for (Fig. 1), while in the absence of these components DnaK exhibits a rather weak affinity for . Thus, these results suggest that under physiological conditions (2-5 mM ATP), DnaJ should modulate the binding activity of DnaK. Such a function has been previously proposed for an eukaryotic DnaJ homologue (Cyr and Douglas, 1994). The regulatory role that DnaJ exhibits on DnaK's substrate binding properties was found to be specific, since we were able to show, using ELISA, that under the same conditions, a mutant DnaJ protein, which lacks the entire 31-amino acid G/F motif, could bind to its protein substrates or DnaK with the same affinity as wild type DnaK, yet was severely defective in regulating DnaK's substrate binding properties (Wall et al., 1995).


Figure 1: DnaJ modulates DnaK's affinity for its substrates. The protein substrates (indicated in the left top corner of each panel) were preincubated (0.5 µg/well) in an ELISA plate as described by Wawrzynow and Zylicz(1995). Following the binding, blocking, and washing procedures, increasing amounts of DnaJ protein in the presence of 50 ng of DnaK (in each well the concentration of DnaK was constant) was incubated without nucleotide (), with ATP (0.5 mM) (bullet), with ADP (0.5 mM) (), with AMP-PCP (0.5 mM) (). After 30 min at room temperature glutaraldehyde was added and the amount of DnaK bound to its different protein substrates was estimated using anti-DnaK serum as described by Wawrzynow and Zylicz(1995).



ADP cannot substitute ATP in DnaJ- and ATP-dependent binding of DnaK to the different protein substrates (Fig. 1). In the case of and O only a slight stimulatory effect of ADP was observed (Fig. 1). When AMP-PCP was used, efficient binding of DnaK to various protein substrates was seen, regardless of whether DnaJ was present or not (Fig. 1). Only in the case of the P and substrates did the presence of ATP and high concentrations of DnaJ stimulate the binding of DnaK to a significantly higher level than the one observed in the absence of DnaJ, but in the presence of AMP-PCP (Fig. 1). This result may suggest that in these cases DnaJ increases the on rate for the DnaK-substrate complex formation. Surprisingly, we also found that in the case of the CMLA substrate, the absence of ATP and the presence of low concentrations of DnaJ partially triggered the release of DnaK from CMLA (Fig. 1). These findings were further substantiated with size exclusion HPLC (Fig. 2). Under these conditions, DnaJ does not interact with CMLA regardless of whether ATP is present or not (Fig. 2). In addition, when ATP was absent, we could not detect, as judged by HPLC, a stable DnaK-DnaJ complex (Wawrzynow and Zylicz, 1995). Nevertheless, DnaJ was able to antagonize the formation of the DnaK-CMLA complex (Fig. 2). These findings are consistent with our ELISA data (Wawrzynow and Zylicz, 1995), suggesting the existence of a weak interaction between DnaK and DnaJ even in the absence of ATP. In parallel experiments, again using the HPLC method, we showed that in the absence of ATP, contrary to the case with CMLA, DnaJ does not release P complexed with DnaK (result not shown). One interpretation of these results is that, when the affinity of DnaK to the substrate is relatively low, DnaJ could be recognized by DnaK as a potential substrate and thus compete with the real one (CMLA).


Figure 2: Isolation of a DnaK-CMLA complex in the presence of DnaJ and ATP. Before injection on a Superdex 200 HPLC column equilibrated with buffer B (Wawrzynow and Zylicz, 1995), DnaK protein (35 µg) was incubated with CMLA (15 µg) in the absence (CMLA, DnaK) or presence of DnaJ (40 µg) (CMLA, DnaK, DnaJ). Control experiments, CMLA alone (CMLA), DnaJ alone (DnaJ), as well as DnaJ preincubated with CMLA (CMLA, DnaJ), are also shown. Each experiment was repeated in the absence (panel A) or presence of 200 µM ATP in the premixture (panel B). The Superdex 200 column was calibrated with the following Bio-Rad molecular weight standards: 1, thyroglobulin (670 kDa); 2, bovine -globulin (158 kDa); 3, chicken ovalbumin (44 kDa); 4, equine myoglobin (17.5 kDa).



When DnaJ and ATP were simultaneously present, permanently denatured CMLA and native P formed a complex with DnaK as judged by either ELISA or HPLC ( Fig. 1and Fig. 2, and results not shown). The SDS-polyacrylamide gel electrophoresis of the fraction after HPLC indicates that DnaJ is not a part of DnaK-P or DnaK-CMLA complexes (results not shown). As shown in Fig. 1and Fig. 2, the efficiency of the DnaJ-dependent activation of DnaK for binding to unfolded CMLA or native P is almost identical. As in the case of P and CMLA, in the presence of DnaJ and ATP it is possible to isolate a stable -DnaK complex (Liberek and Georgopoulos, 1993) and RepA-DnaK complex (Fig. 3). Very likely due to the high affinity of DnaJ for either or RepA (Liberek and Georgopoulos, 1993; Wickner, 1990) in these cases it was possible to isolate tertiary complexes, DnaK--DnaJ (Liberek et al., 1993) or DnaK-RepA-DnaJ (Fig. 3; Wickner et al., 1992; results not shown). Interestingly, in the case of RepA even in the absence of ATP, a tertiary complex could be detected as judged by size exclusion HPLC (Fig. 3) and supported by SDS electrophoresis of fractions after chromatography (results not shown). As reported by Liberek et al.(1992) in the case of , such a tertiary complex in the absence of ATP was not formed. One explanation could be that exhibits relatively high affinity toward DnaJ but low affinity toward DnaK, whereas the RepA protein binds both chaperones equally well (Wawrzynow and Zylicz, 1995).


Figure 3: Isolation of a tertiary DnaK-RepA-DnaJ complex. Before injection on a Superdex 200 column equilibrated with buffer B, the RepA (32 µg) was preincubated with either DnaK (35 µg) (RepA, K), or with DnaJ (40 µg) (RepA, J), or with both DnaK and DnaJ in the absence of ATP (RepA, K, J) or DnaK and DnaJ with ATP (RepA, K, J, ATP) in the premixture. In control experiments RepA was preincubated with DnaK and ATP (200 µM) in the premixture (RepA, K, ATP) and with ATP present both in the premixture and column buffer (RepA, K, ATP/ATP). The positions of RepA and DnaK alone are also shown. The Superdex 200 column was calibrated with the Bio-Rad molecular weight standards described in the the legend to Fig. 2.



To test the stability of the DnaK-P (or DnaK-CMLA) complexes formed in the presence of DnaJ, we preincubated these proteins with ATP for 30 min at room temperature, then washed away the ATP and unbound proteins and added fresh buffer with or without ATP, ADP, AMP-PCP, or ATP with DnaJ, and the reaction was stopped with glutaraldehyde at various times. It was found that ADP and AMP-PCP substantially stabilized the DnaK-P or DnaK-CMLA complexes formed under these conditions (Fig. 4). In the absence of nucleotides the complex was not stable, while the addition of ATP further destabilized these complexes (Fig. 4). Interestingly, the presence of both ATP and DnaJ dramatically stabilized the DnaK-substrate complexes compared to the situation where only ATP was used (Fig. 4). These results suggest that the DnaJ protein may inhibit the off rate of the DnaK-substrate complex.


Figure 4: Stability of the DnaK-substrate complex formed in the presence of DnaJ and ATP. P (panel A) or CMLA (panel B) were preincubated (0.5 µg/well) in ELISA plate wells. The wells were washed and blocked with PBS/BSA and A/BSA as described by Wawrzynow and Zylicz(1995). Subsequently 40 ng of DnaK, 80 ng of DnaJ in buffer A/BSA supplemented with ATP (1 mM), was added to each well and incubation proceeded for 30 min at room temperature, followed by washing twice with 100 µl of buffer A/BSA (time 0). After removal of ATP and unbound proteins, fresh buffer A/BSA was added (50 µl) with: ATP (1 mM), bullet; ADP (1 mM), ; AMP-PCP (1 mM), ; ATP (1 mM) and DnaJ (50 ng), ▴; no nucleotide, . Interacting proteins were cross-linked at the indicated time by the addition of glutaraldehyde (final concentration 0.1%). At each time point, glutaraldehyde cross-linking proceeded for 5 min, followed by washing (three times) with PBS/BSA. The amount of DnaK protein bound to P or CMLA was estimated using an anti-DnaK serum as described by Wawrzynow and Zylicz(1995).



To confirm the requirement of ATP hydrolysis during the DnaJ-dependent formation of DnaK-substrate complex, we preincubated DnaK and DnaJ proteins in the presence of either ATP or ADP or in the absence of any nucleotides for 15 min at 30 °C. Following this, the reaction mixture was immediately added onto an ELISA plate with previously immobilized P protein and the incubation proceeded for an additional 30 min (Fig. 5A). An efficient binding of DnaK to P was observed only when ATP was present during preincubation of DnaK and DnaJ molecular chaperones (Fig. 5A). Neither the presence of ADP nor the absence of any nucleotides promotes DnaJ-dependent binding of DnaK to P (Fig. 5A), suggesting that ATP hydrolysis is required for DnaJ-dependent activation of DnaKs binding to protein substrates. Interestingly, when DnaK, DnaJ, and ATP (or ADP) were preincubated directly on the P-modified ELISA plate wells, similar results were obtained (Fig. 5A), suggesting that the presence of the substrates during DnaJ- and ATP-dependent activation of DnaK is not necessarily required. To test the effect of ADP in the stabilization of P-DnaK complex formed during DnaJ- and ATP-dependent activation of DnaK, we preincubated DnaK, DnaJ, and ATP (or ADP) in an Eppendorf tube (absence of P substrate) for 15 min at 30 °C; following this, a 10-fold molar excess of ADP or ATP was added and the reaction mixture was immediately placed onto an ELISA plate with previously immobilized P protein (Fig. 5B). Surprisingly, when DnaK and DnaJ were preincubated with ATP and added to the substrate in the presence of excess ADP, much more efficient binding of DnaK to the P was observed (Fig. 5B) as compared to the situation where excess of ADP was not used (Fig. 5A). The substitution of ATP by ADP during the first preincubation period (activation reaction) did not lead to efficient binding of the DnaK chaperone (Fig. 5B), thus supporting our previous findings that ATP hydrolysis is required for DnaJ-dependent activation of DnaK protein. The substitution of ADP by ATP during the loading of DnaK and DnaJ onto the substrate also did not lead to an efficient DnaK-P complex formation (Fig. 5B). This observation suggests that ADP is an efficient stabilizing factor of the activated DnaK-P complex. Similar results were obtained when DnaK, DnaJ, and ATP or ADP were preincubated on the ELISA plate with P before the 10-fold molar excess of ADP (or ATP) was added (results not shown). This again may suggest that the presence of substrate during the DnaJ- and ATP-dependent activation of DnaK protein is not required.


Figure 5: ATP hydrolysis is required for the DnaJ-dependent activation of DnaK protein for binding to P. P was preincubated (0.5 µg/well) in ELISA plate wells. The wells were washed and blocked with PBS/BSA and A/BSA as indicated under ``Materials and Methods.'' Panel A, an increasing amount of DnaJ protein in the presence of a constant concentration of DnaK protein (50 ng) was incubated for 45 min directly in the well with ATP (250 µM, bullet), ADP (250 µM, ), or without nucleotide (). The experiment was repeated in such way that an increasing amount of DnaJ protein with DnaK (50 ng) was first preincubated in an Eppendorf tube for 15 min at 30 °C with ATP (), ADP (▴), and no nucleotide (▪). Then, the premixture was loaded on a P-modified ELISA plate and incubation proceeded for additional 30 min at room temperature. The amount of DnaK protein bound to P was estimated as described by Wawrzynow and Zylicz(1995), except no cross-linking with glutaraldehyde was applied. Panel B, an increasing amount of DnaJ protein in the presence of DnaK (50 ng) was first preincubated 15 min at 30 °C in the presence of: ATP (250 µM) ( and bullet) or ADP (250 µM) ( and ▴). Following this, the premixture (50 µl) was loaded on an ELISA plate in the presence of ATP (2.5 mM) ( and ) or ADP (2.5 mM) (bullet and ▴) and incubation at room temperature proceeded for an additional 30 min. The amount of DnaK bound to P was determined using anti-DnaK serum as described by Wawrzynow and Zylicz(1995), except no glutaraldehyde cross-linking was applied. In control experiments, an increasing amount of DnaJ protein with a constant concentration of DnaK was preincubated with ATP (250 µM) directly with P in ELISA wells for 15 min, and after addition of an excess of ADP (2.5 mM), incubation proceeded for additional 30 min.



In a control experiment, following incubation of DnaK or DnaJ with ATP (or ADP) the proteins were separated from unbound nucleotides using a desalting column and directly loaded onto a P-modified ELISA plate. Only in the simultaneous presence of DnaK, DnaJ, and ATP during the preincubation reaction was an efficient binding of DnaK protein to P following size exclusion chromatography observed (results not shown).


DISCUSSION

Using five different protein substrates (including denatured and native protein substrates) we were able to show that in the presence of physiological concentrations of ATP, the affinity of DnaK to its various protein substrates is correlated with the affinity of DnaJ for these protein substrates. The data presented in this paper show that regardless of the low affinity of DnaJ for CMLA (Langer et al., 1992; Wawrzynow and Zylicz, 1995), in the presence of ATP and DnaJ, a DnaK-CMLA complex readily forms. This result was verified by two independent methods, ELISA and size exclusion HPLC. Cyr and Douglas (1994) reported that in the presence of ATP and Ydj1p (an eukaryotic DnaJ homologue) CMLA was released from its complex with Ssa1p (an eukaryotic Hsp70 homologue). It is possible that the prokaryotic and eukaryotic Hsp40/Hsp70 homologues behave differently, even when the same protein substrate is used.

It was previously shown that the presence of DnaJ and GrpE accelerates up to 50-fold the hydrolysis of ATP catalyzed by DnaK (Liberek et al., 1991; Szabo et al., 1994). In addition, ATP hydrolysis is required for the efficient formation of a DnaK-DnaJ complex (Wawrzynow and Zylicz, 1995). In this paper we show that the DnaJ-dependent activation of DnaK to form a stable complex with its various protein substrates also requires ATP hydrolysis. The DnaJ-promoted activation reaction does not occur in the presence of ADP. We propose that the simultaneous presence of DnaJ and ATP hydrolysis converts the conformation of DnaK to such form which binds more efficiently to the different substrates. In agreement with this, recently published data show that the limited trypsin digestion pattern of DnaK in the presence of ATP is different, depending on the presence or absence of DnaJ (Wall et al., 1994).

The mechanism by which DnaJ, in the presence of ATP hydrolysis, induces DnaK's conformational changes remains unclear. Data presented in this paper suggest that after activation of DnaK, ATP could be depleted and the efficient DnaK's binding to the substrate is still observed. We previously found that the DnaK protein possesses an autophosphorylating activity (Zylicz et al., 1983). Sherman and Goldberg(1993) suggested that the phosphorylation of DnaK increases its affinity for a denatured substrate. Formally it is possible that DnaJ, which increases the ATPase activity of the DnaK protein, may also increase the phosphorylation of the DnaK protein. However, recently published data suggest that this is not the case: GrpE, but not DnaJ, influences DnaK's autophosphorylation (Panagiotidis et al., 1994). Another relevant question that remains to be answered is the length of time that DnaK remains in its ``active'' substrate-binding mode, following activation by DnaJ and ATP.

In this work we show that the simple preincubation of DnaK, DnaJ, and ATP (without substrate) is sufficient to activate DnaK to bind to P. This result does not exclude the possibility that in situations when DnaJ alone possesses a high affinity for the protein substrate, e.g. luciferase (Szabo et al., 1994), (Liberek and Georgopoulos, 1993), and RepA (Wickner et al., 1992), DnaJ besides activating DnaK may help by ``presenting'' the protein substrate to DnaK as well. In these cases, the DnaJ-modified substrates in the presence of DnaK and ATP would be converted into a stable tertiary DnaK-substrate-DnaJ complex. In such complexes, the DnaK and DnaJ chaperones may both bind the common protein substrates.

In all other cases we examined, where DnaJ possesses a relatively low affinity toward the substrate (O, P, and CMLA), the presence of DnaJ accelerates the ATP hydrolysis and the formation of a stable substrate-DnaK complex, with which DnaJ interacts only transiently. Recently Liberek et al.(1995) reported that a truncated DnaJ protein (DnaJ12), which lacks the DnaJ peptide binding site but still accelerates DnaK's ATPase activity, can still promote the ATP-dependent formation of a stable DnaK- complex, but itself is not a part of this complex. The possibility that in some cases DnaJ can work catalytically to promote the formation of a DnaK-substrate complex not only does not contradict, but actually is a substantial extension of the model recently proposed by Szabo et al.(1994). In biological terms what our findings imply is that when DnaJ binds tightly to a protein substrate, this may lead to the formation of a DnaK-substrate-DnaJ complex. When DnaJ does not bind efficiently to a substrate, it can still catalyze the formation of the DnaK-substrate complex.

The DnaK-substrate-DnaJ complex is substantially stabilized by ADP. These complexes are destabilized in the absence of nucleotides, and to an even greater extent in the presence of ATP. These results suggest that the induction of conformational changes in DnaK during the release of nucleotides or the exchange of nucleotides to a different form (i.e. ADP to ATP) triggers the release of DnaK from its substrate. Only the presence of ADP, where putative exchange of nucleotides will not induce conformational changes of DnaK, does not destabilize the DnaK-substrate complex. A similar effect of ADP in the stabilization of Hsp70 bound to different substrates has been previously described by other investigators (Prasad et al., 1994; Palleros et al., 1994).


FOOTNOTES

*
This work was supported by Grant 6P203 042 06 from the Polish State Committee for Scientific Research and Swiss National Science Foundation Grant 31-31129.91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: CMLA, carboxymethylated alpha-lactalbumin; AMP-PCP, 5`-adenylyl beta,-imidodiphosphate; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin.


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