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ATP-dependent Proteolysis in Mitochondria

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and them-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.

Molecular chaperone proteins bind non-native protein structures and stabilize them against aggregation (1). By this means, they ensure proper folding of newly synthesized proteins, provide protection against heat denaturation, and mediate the vectorial translocation of polypeptides across biological membranes (2)(3)(4)(5)(6)(7). Furthermore, evidence is accumulating that chaperone proteins play a pivotal role in ATP-dependent proteolytic processes (8 -10). Chaperone and proteolytic activities thereby constitute a quality control system which prevents the possibly deleterious accumulation of misfolded polypeptides in the cell. For instance, the degradation of misfolded polypeptides by Lon-like proteases in Escherichia coli or mitochondria depends on Hsp70 proteins which prevent the aggregation of substrate polypeptides (11)(12)(13)(14)(15). In addition to classical chaperone proteins that cooperate with ATP-dependent proteases during proteolysis, intrinsic chaperone-like properties have been assigned to some ATP-dependent proteases themselves which may be crucial for the degradation of non-native polypep-tides (9,10). The best studied cases are the hetero-oligomeric Clp proteases of prokaryotes whose regulatory subunits exert ATP-dependent chaperone activity (16,17).
Several ATP-dependent proteases have been identified in mitochondria, which mediate the selective degradation of proteins in this organelle (10,18,19). These proteases are required for maintenance of the respiratory competence of yeast cells suggesting important regulatory functions during the biogenesis of mitochondria. An ATP-dependent protease, highly homologous to Escherichia coli Lon protease, has been identified in the mitochondrial matrix space (20,21). The corresponding genes from humans (22,23) and yeast (24,25) were cloned and termed PIM1 (for proteolysis in mitochondria) or LON. PIM1mediated proteolysis is required for the maintenance of mitochondrial genome integrity (24,25) and for the synthesis of mitochondrially encoded subunits of the respiratory chain (70). PIM1 protease forms a high molecular weight, presumably homo-oligomeric complex whose integrity depends on ATP and that mediates the degradation of misfolded polypeptides in the matrix space (26). Proteolysis requires the function of the mitochondrial Hsp70 protein Ssc1p and its co-chaperone Mdj1p, which stabilize substrate polypeptides in a conformation susceptible to degradation by PIM1 protease (15).
Proteins of the inner membrane of mitochondria that are not assembled to functional complexes are degraded by two AAA proteases (27)(28)(29)(30)(31)(32). The subunits of these membrane-bound and metal-dependent proteases harbor characteristic, conserved ATPase domains (33,34). The two known mitochondrial AAA proteases form high molecular weight complexes and expose their catalytic sites to opposite surfaces of the inner membrane: Yme1p, the solely identified subunit of the i-AAA protease, is active on the outer surface (35). In contrast, the catalytic sites of the m-AAA protease, composed of several copies of Yta10p (Afg3p) and Yta12p (Rca1p), are facing the mitochondrial matrix (28,36). The m-AAA protease was found to stably interact with non-native substrate polypeptides suggesting a chaperone-like activity of this protease (28).
In the present manuscript, we report on the requirement of the mitochondrial Hsp70 system for the degradation of misfolded polypeptides by the PIM1 and the m-AAA protease. We exploited the observation that bovine apocytochrome P450scc, missorted to the matrix after import into yeast mitochondria, and a membrane-associated derivative thereof were subject to rapid proteolysis. While P450scc was degraded by PIM1 protease in the matrix, the proteolytic breakdown of the membraneanchored variant was mediated by the m-AAA protease demonstrating overlapping substrate specificities of both proteases. In either case, proteolysis depended on the activity of the molecular chaperones Ssc1p and Mdj1p and on Mge1p, which acts as a nucleotide exchange factor for Ssc1p. Thus, the mtHsp70 system can cooperate with different ATP-dependent proteases in the degradation of non-native polypeptides in mitochondria.

EXPERIMENTAL PROCEDURES
Construction of pSu9(112)-P450scc-Recombinant DNA techniques were applied as described previously (37,38). To generate pSu9(112)-P450scc, a DNA fragment encoding mouse dihydrofolate reductase was excised from Su9(112)-dihydrofolate reductase cloned in pGEM4 (39) by restriction digest with BamHI and HindIII. The HindIII site was filled in with Klenow and the vector was ligated with a BamHI-SmaI fragment of the plasmid pYeDP/coxIV-P450scc (40) encoding mature bovine P450scc lacking the N-terminal 75 residues. The resulting hybrid protein consisted of the 112 N-terminal amino acids of the F 0 -ATPase subunit 9 fused to truncated P450scc.
Yeast Strains and Growth Conditions-Yeast strains carrying mutations in PIM1 were previously described (26). To establish the dependence on the proteolytic activity, PIM1 and a proteolytically inactive variant, PIM1 S1015A , were expressed in ⌬pim1 cells from a multicopy plasmid under the control of a galactose-inducible promotor (26). Cells were grown at 30°C on selective medium containing 2% glucose and 0.5% galactose to induce expression from the GAL1 promotor. Wild type and mutant PIM1 protease were approximately 30-fold overexpressed in these cells (26). Yeast strains carrying mutations in YTA10 or YTA12 are derivatives of W303-1A and are described elsewhere (28). yta10 or yta12 mutant strains were grown on YP medium supplemented with 2% galactose and 0.5% lactate at 30°C according to standard procedures. Yeast strains carrying the temperature-sensitive mutant allele ssc1-3 (41) or mge1-3 (42) were grown at 24°C on selective medium containing 2% galactose and 0.5% lactate. The mdj1-7 mutant strain (43) was grown at 24°C on lactate medium containing galactose instead of glucose.
Import of Preproteins into Isolated Mitochondria and Protein Degradation-Mitochondria were isolated as described previously except that zymolyase treatment was performed at 24°C in the case of temperature-sensitive strains (44,45). pP450scc and pSu9(112)-P450scc were transcribed using SP6 polymerase and synthesized in rabbit reticulocyte lysate in the presence of [ 35 S]methionine (Promega) according to standard procedures (46). Import of the radiolabeled preproteins was performed for 25 min at 25°C essentially as described (15) and halted by the addition of valinomycin (2 M). To allow for proteolysis of newly imported proteins, mitochondria were further incubated in import buffer at 37°C. At the time points indicated, aliquots were withdrawn and diluted 3-fold with ice-cold SHKCl (50 mM HEPES/KOH, pH 7.4, 0.6 M sorbitol, 80 mM KCl). To digest non-imported precursor proteins, samples were treated with trypsin (75 g/ml) for 20 min at 4°C when indicated. Protease digestion was inhibited by adding a 20-fold excess (w/w) of soybean trypsin inhibitor. Mitochondria were then reisolated by centrifugation, washed with SHKCl containing 1 mM phenylmethylsulfonyl fluoride and analyzed by SDS-PAGE. 1 Subfractionation of Mitochondria-The mitochondrial outer membrane was disrupted by osmotic swelling, and mitochondrial proteins were extracted by sodium carbonate pH 11.5 as described previously (36). Subfractionation of mitochondria by digitonin treatment was performed in the presence of proteinase K (50 g/ml) according to published procedures (47).

Bovine Apocytochrome P450scc Is Imported into Yeast Mitochondria, but Is Not Correctly Sorted to the Inner Membrane-
The precursor of bovine apocytochrome P450scc (pP450scc) was synthesized in rabbit reticulocyte lysate in the presence of [ 35 S]methionine and imported into isolated yeast mitochondria in a membrane potential-dependent manner (data not shown). Upon import, the precursor form was converted with low efficiency into a mature form most likely mediated by the mitochondrial processing peptidase (48). Cytochrome P450scc molecules are integral parts of the inner membrane in adrenocortical mitochondria (49). To test the membrane association of newly imported bovine apocytochrome P450scc, yeast mitochondria were treated with carbonate at pH 11.5. This treatment results in the release of soluble proteins into the supernatant fraction, while integral membrane proteins, such as the ADP/ATP carrier, are resistant toward extraction under these conditions. P450scc was almost exclusively recovered from the soluble fraction upon alkaline extraction of the mitochondria, similar to cytochrome b 2 and Mge1p, soluble proteins of the intermembrane and matrix space, respectively (Fig. 1A). Apparently, bovine P450scc is not inserted into the inner membrane of yeast mitochondria. To localize P450scc, yeast mitochondria were treated with increasing amounts of digitonin in the presence of externally added protease. The intermembrane and matrix space became successively accessible to the protease as indicated by the marker proteins cytochrome b 2 (for the intermembrane space) and Mge1p (for the matrix space) (Fig.  1B). Newly imported precursor and mature P450scc were released from mitochondria in parallel with Mge1p indicating that both forms are localized in the mitochondrial matrix (Fig.  1B). These results demonstrate that bovine apocytochrome P450scc is imported into yeast mitochondria and, although 1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid .

FIG. 1. Localization of bovine pP450scc in yeast mitochondria.
A, subfractionation of mitochondria by alkaline extraction. Newly synthesized, [ 35 S]methionine-labeled pP450scc was imported into isolated yeast mitochondria for 25 min at 25°C, and non-imported preproteins were digested with trypsin as described under "Experimental Procedures." Mitochondria were then resuspended in 0.1 M Na 2 CO 3 , pH 11.5, and 1 mM phenylmethylsulfonyl fluoride and separated into soluble and pellet fractions by centrifugation as described previously (36). Protein fractions were analyzed by SDS-PAGE, followed by autoradiography and immunoblotting using polyclonal antisera directed against cytochrome b 2 (cyt b 2 ), the ADP/ATP carrier (AAC), and Mge1p. T, total; P, pellet fraction; S, supernatant fraction. B, digitonin fractionation of mitochondria. After import of pP450scc and trypsin digestion, mitochondria were treated with increasing amounts of digitonin in the presence of proteinase K. After centrifugation, proteins in the pellet fraction were analyzed by SDS-PAGE and blotted onto nitrocellulose. Immunoblotting of the endogenous marker proteins cytochrome b 2 (Cyt b 2 ) (E) and Mge1p (q) was performed using a chemiluminescence detection kit and quantified by laser densitometry. The amount of the protein in samples incubated with proteinase K in the absence of digitonin was set to 100%. OE, pP450scc, precursor form of P450scc; ‚, mP450scc, mature form of P450scc.
with low efficiency, processed to the mature form. In contrast to adrenocortical mitochondria, however, P450scc molecules are not inserted into the inner membrane but accumulate in the matrix space. Thus, intramitochondrial sorting appears to depend on a specific sorting pathway in adrenocortical mitochondria or on the lipid composition of the membrane. It should be noted, however, that at least a fraction of P450scc appears to be correctly inserted into the inner mitochondrial membrane after expression in Saccharomyces cerevisiae, as submitochondrial particles exhibited cholesterol hydroxylase activity in the presence of externally added adrenodoxin and adrenodoxin reductase (40).
ATP-dependent Degradation of P450scc by PIM1 Protease in the Matrix-The stability of newly imported, missorted apocytochrome P450scc was analyzed in further experiments. After import of P450scc, the electrochemical gradient across the mitochondrial inner membrane was dissipated by adding the uncoupler valinomycin, and then mitochondria were incubated at 37°C. Newly imported P450scc was degraded in mitochondria in a time-dependent fashion (Fig. 2). Proteolysis was impaired upon depletion of mitochondrial matrix ATP (Fig. 2).
To identify the protease responsible for the proteolytic breakdown of P450scc, mitochondria were isolated from yeast strains carrying mutations in either the PIM1 or the m-AAA protease, and the stability of newly imported P450scc was examined. Inactivation of either protease does not affect the accumulation of the other protease in mitochondria excluding indirect effects of the mutations (data not shown). Proteolysis of P450scc was impaired in mitochondria lacking PIM1 protease (Fig. 3A). Impairment of proteolysis of P450scc might result from a disturbed energy metabolism in pim1 mutant cells, which accumulate lesions in mtDNA and therefore are respiratory-deficient (24,25). To exclude this possibility, the stability of P450scc was examined in ⌬pim1 mitochondria which lack mtDNA but contain PIM1 protease. Wild type and the proteolytically inactive variant PIM1 S1015A protease carrying a mutation in the proteolytic center (26) were expressed in ⌬pim1 cells. Newly imported P450scc was degraded, although with reduced efficiency, in ⌬pim1 mitochondria harboring the wild type form of the protease, while proteolysis was not restored upon expression of PIM1 S1015A protease (Fig. 3A). Thus, degradation of P450scc depends on the proteolytic activity of PIM1. In contrast, degradation of P450scc was not significantly affected by mutations in the catalytic sites of Yta10p (E559Q) or Yta12p (E614Q), which are subunits of the membrane-bound m-AAA protease (Fig. 3B). Both mutations have previously been observed to abolish the proteolytic breakdown of nonassembled polypeptides in the inner membrane by the m-AAA protease (27,28). We conclude from these experiments that missorted P450scc is degraded by PIM1 protease in the mitochondrial matrix space.
Missorted P450scc Is Prone to Aggregation in Mitochondria-Newly imported P450scc, missorted to the mitochondrial matrix, most likely cannot attain its native conformation. It is therefore conceivable that P450scc is prone to aggregation in yeast mitochondria. P450scc, however, was recovered in the soluble fraction of mitochondria, even when these were incubated at 37°C for 15 min (Fig. 4). Likewise, the solubility of P450scc was not affected after import into mitochondria lacking Yta10p or Yta12p and therefore m-AAA protease activity (Fig. 4). In contrast, P450scc formed insoluble aggregates in mitochondria lacking PIM1 protease (Fig. 4). These results demonstrate that missorted P450scc is prone to aggregation in yeast mitochondria and further substantiate the requirement of PIM1 protease for the proteolytic breakdown of P450scc.
The aggregation of P450scc in the absence of PIM1 might reflect a deficiency in a chaperone-like function of the protease, which has recently been proposed to exist independent of its proteolytic activity (50). Therefore, proteolytically inactive PIM1 S1015A was overexpressed in pim1 null mutant cells, and aggregation of newly imported P450scc was analyzed (Fig. 4). Replacement of the conserved serine 1015 by alanine abolishes proteolytic activity of PIM1 but does not affect the overall protein stability nor the ATP-dependent assembly of the pro-  3. Degradation of P450scc depends on the presence of a proteolytically active PIM1 protease in mitochondria but is not affected by mutations in m-AAA protease subunits. pP450scc was imported into mitochondria isolated from a ⌬pim1 strain (Ⅺ) and from ⌬pim1 strains expressing PIM1 (⌬pim1/PIM1)(E) or PIM1 S1015A (pim1 S1015A )(f) (A) or from wild type cells (WT)(E), from a ⌬yta10 strain expressing YTA10 E559Q (yta10 E559Q )(Ⅺ), and from a ⌬yta12 strain expressing YTA12 E614Q (yta12 E614Q )(f) (B). After inhibition of import by adding valinomycin, samples were further incubated at 37°C in the presence of an ATP-regenerating system and analyzed as described in Fig. 2. The total amount of imported mature and precursor forms of P450scc prior to incubation at 37°C was set to 100%. tease subunits (26,50). PIM1 S1015A protease did not stabilize P450scc against aggregation (Fig. 4), although it was present at an approximately 30-fold higher level in mitochondria when compared with wild type cells (26). Apparently, PIM1 protease on its own is not capable of maintaining missorted P450scc in a soluble state.
Requirement of the mtHsp70 System for the PIM1-mediated Degradation of P450scc-To examine a possible role of the mitochondrial Hsp70 protein Ssc1p in preventing aggregation of P450scc, we used mitochondria from the ssc1-3 yeast strain containing a temperature-sensitive mutant form of Ssc1p (41). Furthermore, the stability of P450scc was analyzed in mitochondria carrying a conditional mutant form of Mdj1p (mdj1-7) (43), which also seems to act as a molecular chaperone in mitochondria and ensures efficient binding to and release from Ssc1p (15,51). After import of pP450scc into mitochondria at permissive temperature, samples were incubated at 37°C to inactivate the mutant forms of the proteins and to allow degradation to occur. While P450scc was degraded in wild type mitochondria, proteolysis was impaired in ssc1-3 and mdj1-7 mutant mitochondria at non-permissive temperatures (Fig.  5A). P450scc formed insoluble aggregates under these conditions (Fig. 5B). Thus, similar to other misfolded proteins, the degradation of P450scc depends on Ssc1p and Mdj1p which stabilize P450scc molecules against aggregation and thereby allow their proteolytic breakdown by PIM1.
Polypeptide binding to Ssc1p is also regulated by Mge1p, which acts as a nucleotide exchange factor for Ssc1p (42,(52)(53)(54). A possible role of Mge1p during proteolysis of misfolded polypeptides in mitochondria, however, has not been reported until now. The degradation of P450scc was therefore analyzed in mitochondria carrying the temperature-sensitive mutant allele mge1-3 (42). Inactivation of Mge1-3p at non-permissive temperature resulted in the stabilization of newly imported P450scc (Fig. 5A). In contrast to ssc1-3 or mdj1-7 mitochondria, P450scc accumulated in a soluble form in mge1-3 mitochondria (Fig. 5B), most likely due to an impaired release of P450scc from Ssc1p (52). These results establish a crucial function of all components of the mtHsp70 system during the PIM1mediated proteolysis of misfolded proteins in mitochondria.
An Apocytochrome P450scc Variant with an Amino-terminal Transmembrane Region Is Anchored to the Inner Membrane-To ensure sorting of bovine apocytochrome P450scc to the mitochondrial inner membrane, the targeting sequence and the amino-terminal 75 amino acid residues of P450scc were replaced by the first 112 amino acids of the subunit 9 of the F 1 F 0 -ATPase of Neurospora crassa. This sequence contains the mitochondrial targeting sequence and the first transmembrane region of ATPase subunit 9 and has previously been used to target heterologous proteins to the inner membrane (55). Import of the resulting hybrid protein, Su9(112)-P450scc, depended on an electrochemical potential across the mitochondrial inner membrane (Fig. 6A). To determine the submitochondrial localization of Su9(112)-P450scc, mitochondria were fractionated by osmotic swelling (Fig. 6B). Su9(112)-P450scc was not released into the supernatant upon disruption of the outer membrane nor degraded by protease which was externally added to mitoplasts suggesting its presence in an internal mitochondrial compartment (Fig. 6B). The association of Su9(112)-P450scc with the inner membrane was examined by treatment of mitochondria with carbonate at pH 11.5 (Fig.  6B). In contrast to P450scc, the majority of newly imported Su9(112)-P450scc was recovered from the pellet fraction after extraction of mitochondria indicating the insertion of the protein into the inner membrane. In agreement with these findings, Su9(112)-P450scc became solubilized in parallel with the ADP/ATP carrier when mitochondria were treated with increasing amounts of digitonin (data not shown). We conclude from these experiments that Su9(112)-P450scc is tightly associated with the inner membrane.
Membrane-anchored P450scc Is Degraded by the m-AAA Pro- FIG. 5. PIM1-mediated proteolysis of newly imported P450scc depends on the mtHsp70 system. pP450scc was imported into wild type (WT) (q), ssc1-3 (‚), mdj1-7 (E), and mge1-3 (OE) mutant mitochondria for 25 min at 25°C as described under "Experimental Procedures." A, degradation of P450scc is impaired in ssc1-3, mdj1-7, and mge1-3 mutant mitochondria at non-permissive temperature. After import at permissive temperature, samples were further incubated at 37°C, and the stability of P450scc was analyzed as described in Fig. 2. B, newly imported P450scc aggregates in ssc1-3 and mdj1-7, but not in mge1-3 mutant mitochondria at non-permissive temperature. After completion of import, mitochondria were incubated for 15 min at 37°C. Aggregation of P450scc was analyzed as described in Fig. 3C. tease-Sonication of adrenocortical mitochondria in the presence of trypsin results in the clipping of endogenous cytochrome P450scc in two fragments of 26 and 29 kDa, which is considered to indicate a native conformation of the protein in the inner membrane (56,57). Newly imported Su9(112)-P450scc, however, was completely degraded by trypsin when mitochondria were lysed by sonication (data not shown). Although attached to the inner membrane, the P450scc moiety of the hybrid protein apparently does not fold into its native conformation and is most likely exposed to the matrix. Therefore, the stability of Su9(112)-P450scc in mitochondria was examined in further experiments. The membrane-anchored fusion protein was degraded in wild type mitochondria (Fig. 7A). Su9(112)-P450scc was, however, stabilized in mitochondria lacking proteolytically active m-AAA protease: proteolysis was impaired in ⌬yta10 and ⌬yta12 mitochondria expressing proteolytically inactive forms of either subunit of the m-AAA protease, Yta10 E559Q p or Yta12 E614Q p (Fig. 7A). In contrast, the proteolytic breakdown of Su9(112)-P450scc occurred with similar kinetics in ⌬pim1 mitochondria expressing either the wild type or the proteolytically inactive form of the PIM1 protease (Fig. 7B). Thus, membrane-anchored Su9(112)-P450scc, in contrast to matrix-located P450scc, is degraded by the m-AAA protease.
Degradation of Membrane-anchored P450scc by the m-AAA Protease Depends on the mtHsp70 System-It is conceivable that molecular chaperones are required to maintain the matrix-exposed domain of Su9(112)-P450scc in a conformation susceptible to degradation by the m-AAA protease. To analyze the involvement of the mtHsp70 system in the proteolysis of membrane-anchored P450scc, Su9(112)-P450scc was imported at permissive temperature into ssc1-3, mdj1-7, and mge1-3 mutant mitochondria. These conditions allow import of Su9(112)-P450scc into mitochondria and its insertion into the inner membrane. The stability of newly imported, membraneassociated Su9(112)-P450scc was then examined at non-permissive temperature (Fig. 8). While Su9(112)-P450scc was degraded in wild type mitochondria, proteolysis was impaired upon inactivation of Ssc1p, Mdj1p, or Mge1p in ssc1-3, mdj1-7, or mge1-3 mitochondria, respectively. These results demonstrate that degradation of membrane-bound Su9(112)-P450scc by the m-AAA protease depends on Ssc1p and the co-chaperones Mdj1p and Mge1p which act after insertion of the polypeptide into the inner membrane. DISCUSSION Molecular chaperone proteins fulfill essential functions during the proteolysis of misfolded polypeptides in mitochondria. The mtHsp70 system cooperates not only with PIM1 protease in the degradation of non-native polypeptides accumulating in the matrix, but in addition is required for the proteolytic breakdown of at least some polypeptides which are associated with the inner membrane and degraded by the m-AAA protease. The proteolysis of misfolded polypeptides depends on the activities of Ssc1p and Mdj1p, which both exert chaperone activity and thereby most likely prevent the irreversible misfolding of polypeptides prone to degradation. Moreover, we assign a crucial role for the proteolysis of misfolded proteins to Mge1p, which acts as a nucleotide exchange factor of Ssc1p.  (112)-P450scc for 20 min at 25°C, the outer membrane was disrupted by osmotic swelling in the presence or absence of proteinase K (20 g/ ml)(PK) as indicated. In parallel, mitochondria were extracted with 0.1 M Na 2 CO 3 , pH 11.5, and split into soluble and pellet fractions by centrifugation as in Fig. 1. Proteins in both fractions were separated by SDS-PAGE, blotted onto nitrocellulose, and analyzed by autoradiography and immunoblotting using cytochrome b 2 (cyt b 2 ), the ADP/ATPcarrier (AAC), and Mge1p as marker proteins.
The mtHsp70 system is required for both folding and degradation of mitochondrial proteins and thereby comprises a quality control system in mitochondria. The experiments described in the present manuscript, together with previous findings (8,9,15,19), suggest that folding-competent polypeptides are distinguished from irreversibly misfolded polypeptides by kinetic partitioning. Newly imported preproteins attain their native state in the matrix upon cycles of binding to and release from Ssc1p. Folding occurs upon release from the chaperone in the unbound state or in association with the mitochondrial Hsp60 machinery (6,58,59). The ATP-dependent binding of a polypeptide to Ssc1p is regulated by Mdj1p and Mge1p. Irreversibly misfolded polypeptides undergo additional binding and release cycles and remain associated with the mtHsp70 system for a prolonged time. This may allow the recognition of the polypeptides by the protease and, after release from the chaperone protein, their degradation. A role of the mtHsp70 system for the folding of inner membrane proteins remains to be examined; however, our results suggest that soluble and at least some membrane-embedded proteins are affected similarly by chaperone proteins. The functional cooperation of the mtHsp70 system with two different ATP-dependent proteases suggests that no specific recognition of chaperone proteins by the proteases occurs and is therefore in agreement with a kinetic partitioning of substrate polypeptides between chaperone proteins and proteases.
The function of Mge1p is essential for the degradation of misfolded proteins by different proteases in mitochondria, most likely as it modulates their binding to Ssc1p (42,51,52,60). While polypeptides prone to degradation form aggregates if the activity of the chaperone proteins Ssc1p and Mdj1p is impaired, they accumulate in the soluble state in the absence of Mge1p function. Substrate polypeptides apparently are not released from the chaperone proteins under these conditions. Mge1p promotes the release of ADP from Ssc1p allowing the binding of ATP to the chaperone (52)(53)(54). Upon ATP binding, substrate polypeptides dissociate from Hsp70 proteins (61)(62)(63). Thus, impaired nucleotide release from Ssc1p in the absence of Mge1p inhibits the release of associated substrate polypeptides, a prerequisite for their degradation.
The m-AAA protease stably binds non-native, integral membrane proteins which are prone to degradation suggesting chaperone-like properties of the protease (28). A chaperone-like activity of the protease might be needed to stabilize polypeptides in a conformation susceptible to proteolysis. This might include unfolding of hydrophilic domains or the extraction of transmembrane segments from the lipid bilayer. Nevertheless, degradation of at least some membrane-associated polypeptides by the m-AAA protease depends, in addition, on the mtHsp70 system. This finding is reminiscent of studies in E.
coli which demonstrate the requirement of the DnaK system for the degradation of the soluble transcription factor 32 by FtsH, a homologue of the mitochondrial m-AAA protease (64 -67). It is conceivable that molecular chaperones are specifically required to prevent misfolding of large, solvent-exposed domains of membrane-associated polypeptides, while proteolysis of other membrane proteins by the m-AAA protease is independent of the mtHsp70 system. Indeed, the proteolytic breakdown by the m-AAA protease of non-assembled, mitochondrially encoded subunits of respiratory chain complexes, which lack large solvent-exposed domains, was not affected in ssc1-3 mutant mitochondria at non-permissive temperature. 2 Our results demonstrate an overlapping substrate specificity of the m-AAA protease with the PIM1 protease. A misfolded polypeptide is degraded by either protease depending on its localization in mitochondria. This finding is in agreement with genetic evidence indicating functional similarities between both proteases (50, 68). Next to nothing is known about the sequence specificity of ATP-dependent proteases in mitochondria. Studies on the E. coli Lon protease, however, suggest a rather degenerate specificity of Lon-like proteases (69). Similarly, the analysis of proteolytic fragments of a model protein generated by the i-and m-AAA protease did not reveal a conserved cleavage motif (35). The degradation of mitochondrial proteins by ATP-dependent proteases is apparently mainly initiated by a non-native conformation of the polypeptide. Molecular chaperones and chaperone-like properties of the proteases may therefore play a crucial role in the recognition of substrate polypeptides.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Direct Measurement of the Substrate Preference of Uracil-DNA Glycosylase*

Site-directed mutants of the herpes simplex virus type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373, 487–493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87–92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis. The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single- and double-stranded oligonucleotides, with or without uracil, was monitored by real-time biomolecular interaction analysis using surface plasmon resonance. Both mutants exhibited comparable rates of binding and dissociation on the same uracil-containing substrates. Interaction with single-stranded uracil-DNA was found to be stronger than with double-stranded uracil-DNA, and the binding to Gua:Ura mismatches was significantly stronger than that to Ade:Ura base pairs suggesting that the stability of the base pair determines the efficiency of interaction. Also, there was negligible interaction between the mutants and single- or double-stranded DNA lacking uracil, or with DNA containing abasic sites. These results suggest that it is uracil in the DNA, rather than DNA itself, that is recognized by the uracil-DNA glycosylases.

Uracil-DNA glycosylases (UDG) 1 are a highly conserved and ubiquitous class of DNA-repair enzymes that catalyze the excision of uracil bases from DNA (1). Uracil is an RNA base and does not normally occur in DNA, though it forms a good Watson-Crick base pair with adenine. Uracil and thymine differ in that thymine is methylated at the 5-position of the pyrimidine ring, and this is not involved in forming the base pair. Uracil can occur in DNA either by misincorporation of deoxyuridine triphosphate during replication by DNA polymerases, which apparently do not discriminate between this nucleotide and thymidine triphosphate, or as the product of spontaneous hydrolytic deamination of cytosine residues in DNA (1,2). The Gua:Ura mismatches that result from cytosine deamination are promutagenic, leading to Gua:Cyt 3 Ade:Thy transition mutations unless the uracil is repaired to the original cytosine (1,2). As thymine methyls in Ade:Thy base pairs are essential in sequence-specific recognition by many regulatory DNA-binding proteins (3), Ade:Ura base pairs, although not mutagenic, are potentially disruptive.
Recently, the structural basis for the exquisite recognition of uracil by UDGs has been elucidated (4 -6). The structures show that these enzymes are able to accomodate sequence nonspecific DNA along a channel and that this channel contains an active site pocket that is perfectly tailored to admit only uracil bases. The evidence for a DNA-binding channel is further supported by the structures of a specific peptide inhibitor of the UDGs in complex with the enzymes. This inhibitor prevents binding of UDG to DNA, and is seen to mimic a polynucleotide in its interaction with the enzyme (7,8).
The structures of DNA modification and repair enzymes in complex with polynucleotides either show directly, or indicate the likelihood of, the extrusion of the target base from the DNA duplex (6, 9 -11). In the case of the UDGs, there is no apparent facility for a gross conformational change in the enzyme, and the only way that uracil can enter into the specific pocket is for the base to become extrahelical. This has recently been shown structurally for UDG (6).
The phenomenon of "base-flipping" has yet to be experimentally deconvoluted from the reaction as a whole, and it is still open to debate as to whether the extrusion of DNA bases from a duplex is spontaneous or is actively promoted by the DNA modification or repair enzymes (12). Structural analysis alone is unable to distinguish the two possible mechanisms. Mutation of active site residues to yield catalytically inactive mutant proteins coupled with careful binding studies may well yield a satisfactory answer to this question; it is with this question in mind that these studies were undertaken. Both of the residues implicated in catalysis by structural studies, aspartic acid-88 and histidine-210, were mutated to asparagine and used in binding studies to a range of oligonucleotides using surface plasmon resonance. This system is a powerful means of attempting to deconvolute the mode of substrate recognition and binding by UDG in the absence of catalysis.

Construction of Recombinant Mutants of HSV1 UDG-Aspartic
acid-88 and histidine-210 of the recombinant HSV1 UDG were both mutated to asparagine (D88N and H210N, respectively) to abolish catalytic activity. Oligonucleotide 18-mers were designed to change the codons in the open reading frame from aspartic acid and histidine, * This work was supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom correspondence should be addressed. respectively, to asparagine with the substitution of just one nucleotide in each case. The oligonucleotides were designed such that the mismatch was located in the middle of the sequence. The method used for mutagenesis was the Altered Sites II system (Promega) and was followed as per protocol. The mutagenesis was effected by subcloning the HSV1 UDG open reading frame from the original expression vector pTS106⅐ (13) using NcoI and filling the overhang with Klenow purification of DNA by Geneclean (Anachem) and final excision of the open reading frame using HindIII, and inserting it into the mutagenesis vector supplied with the kit (pALTER1) into SmaI and HindIII, thus recreating the NcoI site for subsequent recloning into pTrc99A following mutagenesis. Unique restriction sites were introduced by mutagenesis (EcoO109I for D88N, and BstBI for H210N), and these were used to screen for positive mutant DNAs. Positively identified mutants were cloned as NcoI to HindIII fragments in pTrc99A (Pharmacia Biotech Inc.) cut with the same enzymes.
Expression and Purification of Mutant Proteins-Expression was performed in the Escherichia coli strain BL21(DE3), and standard assays were carried out for UDG activity (14). Inactive mutant clones were stored as 50% glycerol stocks at Ϫ80°C. Expression of D88N mutant resulted in low level expression of insoluble protein under the standard expression conditions (13), and optimum expression was obtained by growing the bacteria to A 600 nm ϭ 0.8 -1.0 at 28°C, before inducing with 0.5 mM isopropyl-␤-D thiogalactopyranoside for 36 h. The H210N mutant was expressed under the standard conditions (13). Purification was by the following method. Following lysis of cells and partition of soluble and insoluble fractions, (13), the clarified supernatant was applied to a two-column system (60 ml Q-Sepharose outlet into the inlet of 60 ml SP-Sepharose) equilibrated with 20 mM Tris-HCl, 10 mM EDTA, pH 8.3, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM dithiothreitol chilled in an ice-bath. A wash of 800 ml of the equilibration buffer was performed subsequent to loading, and a further 400 ml wash using the same buffer was then performed on the SP-Sepharose column alone. A 0 to 1.6 M sodium chloride gradient was run through the SP-sepaharose column over 7 column volumes. Fractions containing the bulk of mutant UDG, as judged by SDS-polyacrylamide gel electrophoresis analysis, were pooled and diluted such that the final sodium chloride concentration was less than 50 mM, using buffer B (20 mM Tris-HCl/10 mM EDTA pH 8.3, 10% glycerol, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM dithiothreitol). The diluted protein was loaded onto a 25-ml poly-U-Sepharose column, pre-equilibrated in buffer B. A wash of 5-column volumes of buffer B with 100 mM sodium chloride was performed after loading, and then a gradient of 100 -250 mM sodium chloride over 5-column volumes was performed. UDG mutant protein was eluted in a 2 M sodium chloride step. Dialysis was performed twice against 100 volumes of 10 mM Tris-HCl, pH 8.3, 0.1 mM phenylmethanesulfonyl fluoride. The proteins were shown to be greater than 95% pure after this procedure, with D88N having a final yield of 2 mg/liter bacterial culture and with H210N having a final yield of 15 mg/liter bacterial culture.
Crystallization of Mutant UDG Proteins-Proteins were concentrated following purification in Amicon-stirred ultrafiltration cells using PM10 membranes (Amicon Corp.). The D88N mutant was far less soluble than H210N, and was used for crystallization trials at 15 mg/ml. The H210N mutant was used at 35 mg/ml. Purified concentrated proteins were 0.22-mm filtered in Ultrafree MC microcentrifuge cups (Millipore), and phenylmethanesulfonyl fluoride and sodium azide were added to final concentrations of 0.1 mM and 0.02% (w/v), respectively. The protein stocks were then stored at 4°C where they were stable for several months. Both mutants were put through crystallization trials using the Hampton research crystal screen, implemented as 1:1 microbatch mixtures under paraffin oil in Terazaki plates (15). Plates were stored at 16°C in the dark. Similar screens were also performed for proteins mixed at ratios of 1:2 (protein:ligand) with nucleotides and oligonucleotides.
Preparation of Immobilized Ugi Protein-The Ugi protein was prepared as described previously (16), and immobilized on Affi-Gel 15 cationic beads (Bio-Rad) using the following protocol. Beads, stored as supplied at Ϫ20°C, were allowed to reach room temperature over 30 min with occasional inversion of the bottle. After thoroughly resuspending the beads by inversion and agitation, a volume of approximately 120 l was withdrawn with a 1-ml micropipettor using a wide bore tip (made by sawing off one-third of the tapered end) and transferred to a 1.5 ml microcentrifuge tube. The tube was pulsed for 30 s at 16,000 ϫ g to pellet the beads, and the bulk of the isopropanol-rich medium was discarded. A volume of 500 l of ice-cold deionized water was added to the beads, and the tube was vortexed for 5 s. The tube was pulsed as described, and the water was discarded. This was repeated twice more, and then finally with 50 mM HEPES-NaOH, pH 7.0. Upon discarding the HEPES-NaOH, 400 l of HEPES-NaOH, pH 7.0, containing 3 mg of purified Ugi protein was added to the beads. The tube was agitated gently to suspend the beads in the protein solution, and the agitation was continued on a shaker for 90 min at room temperature. The protein binds to the beads through lysine ⑀-amino groups during this step. The tube was pulsed as described previously, and the supernatant was removed and stored. The beads were washed three times with 500 l of 50 mM HEPES-NaOH, pH 7.0, as described earlier, with the supernatant being stored after each spin. After removing the supernatant following the final spin, 10 l of freshly prepared ethanolamine, pH 8.0, were added and mixed in by tapping the tube gently; the tube was allowed to stand for an hour at room temperature. This step blocks any beads that have not bound protein. The tube was pulsed as described previously, discarding the supernatant, and was washed twice with 400 l of 50 mM HEPES-NaOH, pH 7.0, discarding the supernatant each time. The beads were then ready to use.
Synthesis and Purification of Oligonucleotides Used in These Binding Studies-The oligonucleotide sequence used in these experiments was 5Ј-biotin-CCGAATCAGTTCACTTCNAGCCGAGGTATTTAGCC, for oligonucleotides ssC and ssU, where N is C in ssC and N is U in ssU. The nonbiotinylated oligonucleotides were 5Ј-GGCTAAATACCTCGGCT-NGAAGTGAACTGATTCGG where N is either A or G to form the duplexes dsU/A and dsU/G, respectively, with the biotinylated strand ssU, and the duplex dsC/G with ssC. Oligonucleotide synthesis was performed on an Applied Biosystems 394 DNA synthesizer on the 0.2-m scale using cyanoethyl phosphoramidite chemistry. Standard DNA synthesis reagents and cyanoethyl phosphoramidite monomers were obtained from Applied Biosystems, Ltd. The biotin phosphoramidite was obtained from Cruachem Ltd. and used as a 0.15 M solution in anhydrous acetonitrile; the coupling time was extended to 3 min.
Stepwise coupling efficiencies were measured automatically on the synthesizer by trityl analysis, and all monomers were coupled at greater than 98%. Oligonucleotides were deprotected in concentrated aqueous ammonia for 8 h at 55°C. High performance liquid chromatography purification was carried out on a Gilson model 306 high performance liquid chromatography system using a Brownlee Aquapore octyl reverse phase column (10 ϫ 250 mm) with a flow rate of 3 ml/min and a gradient of 0 -75% buffer B over a period of 30 min. (Buffer A, 0.1 M triethylammonium acetate; buffer B, 0.1 M triethylammonium acetate with 25% acetonitrile). In the case of oligonucleotides labeled with biotin, the correct product was the major peak (final peak to elute from the column). After high pressure liquid chromatography purification, the major product was evaporated to dryness and desalted using a Pharmacia NAP 10 column (Sephadex G25) according to the manufacturer's instructions.
Experimental Assay Procedure Using Surface Plasmon Resonance-The principle of operation of the BIAcore biosensor and its use in analyzing protein-DNA interactions have been described before (17,21). All interactions were analyzed in binding buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) at a constant flow rate of 5 l/min and at a constant temperature of 25°C. Biotinylated oligonucleotides were injected over a streptavidin-coated sensor chip (SA5, Pharmacia Biosensor) until a suitable level was achieved (see "Results"). For the formation of double-stranded oligonucleotides, nonbiotinylated DNA was injected until no more increase in binding to the immobilized single-stranded DNA could be observed. Bound protein was eluted from the DNA by a short pulse (5 l) of 0.05% SDS. This regeneration procedure did not alter to any measurable extent the ability of the immobilized DNA to bind protein in subsequent cycles.
Analysis of the data was performed using the evaluation software supplied with the instrument. To eliminate small "bulk" refractive change differences at the beginning and end of each injection (due to small differences in buffer composition of the stock protein solutions), a control sensorgram obtained over a nonbinding surface was subtracted for each protein injection. For obtaining the association and dissociation rate constants the following equations were used, respectively.
R, R 0 , and R eq are the response at time t, t 0 , and at equilibrium, respectively; C, the concentration of protein; k a , the association rate constant; k d , the dissociation rate constant; and t 0 , the start time of the dissociation or association. For the determination of the equilibrium dissociation constant (K D ) from binding experiments was used (where R max is the maximum response level).

Confirmation of Tertiary Structure of Mutant Proteins-
The integrity of the tertiary structures of the mutant proteins was verified in the absence of catalytic activity against a standard substrate routinely used to assay UDGs (14) by the following methods. First, Bacillus subtilis bacteriophage PBS1 Ugi protein (16) immobilized on Affi-Gel 15 beads, was shown by SDS-polyacrylamide gel electrophoresis to sequester and by standard assay (14) to inactivate wild-type HSV1 UDG. Similarly immobilized Ugi protein was shown by SDS-polyacrylamide gel electrophoresis to sequester both mutants with similar affinity to its capture of wild-type enzyme. To remove the UDG or mutants from the Ugi protein, it was necessary to heat the beads in the presence of SDS. As a control, whole protein from an E. coli whole cell lysate was shown by SDS-polyacrylamide gel electrophoresis to bind very weakly apart from E. coli UDG, which was retained strongly. This strongly suggests that the DNA-binding channel and overall fold of the mutant proteins do not significantly differ from that of the wild-type enzyme.
The second method used to indicate correct folding was using crystallization and x-ray diffraction. Both D88N and H210N mutants were crystallized using the Hampton research crystal screen following purification, and the latter also formed rectangular rod-like crystals spontaneously in situ after 10 days at 4°C, average size 80 mm thick by 200 mm long, which diffracted to 2.5 Å on a laboratory source. The spacegroup was found to be P2 1 with unit cell a ϭ 42.9 Å, b ϭ 61.6 Å, c ϭ 43.9 Å, and ␤ ϭ 93.05, with a solvent content of 58% and 1 molecule in the asymmetric unit. Due to the fact that this is a monoclinic crystal form, a complete dataset has not yet been collected, and a structure is not yet available. The needle-like crystals of the D88N and H210N mutants obtained in crystal screens from polyethylene glycol 4000/buffer mixtures were too small for x-ray analysis, but co-crystals of the D88N mutant with the single-stranded trideoxynucleotide pdTpdTpdU, resulted in six-sided plates with average dimensions of 100-mm across by 20 -50-mm thick. These crystals are seen to diffract beyond 2.6 Å at a synchrotron radiation source. Further characterization is in progress. The D88N mutant also yields rod-like crystals with the nucleotides 3Ј-dUMP and 5Ј-dUMP, and both mutants yield large orthorhombic rods with the trideoxynucleotide pdT-pdTpdT identical to those obtained with the wild-type enzyme. Crystals of macromolecules, which diffract to medium and high resolution, are a good indication that there is inherent order and homogeneity in the components of the lattice. Morphological changes of protein crystals in the presence of potential ligands is a good indication that productive binding has taken place, demonstrating that the protein is correctly folded.
Binding of Proteins to Immobilized Oligonucleotides-The BIAcore biosensor was used to measure the specificity and affinity of the interactions between mutant or wild-type enzyme and single-or double-stranded oligonucleotides. In this system, the biotinylated oligonucleotide is immobilized on a streptavidin-coated dextran layer attached to a sensorchip, and the protein is injected at a constant flow rate. The biosensor measures refractive index changes close to the dextran layer, which in turn correspond to changes in the amount of protein bound to the surface. The plot of bound protein (measured in arbitrary resonance units (RU)) versus time is called a sensorgram and can be used to derive kinetic or equilibrium constants. This method has been used before to study other pro-tein-DNA interactions (17)(18)(19). To immobilize double-stranded oligonucleotides, one of the strands was biotinylated and attached to the surface first and then the other strand was injected at saturating amounts until no further binding was observed (see "Experimental Procedures"). Complete formation of double-stranded DNA was verified by observing approximate doubling of the static resonance signal after the annealing of the second strand (Fig. 2). There was no measurable dissociation of DNA from the surface even after many cycles of binding and regeneration. Measurements of protein-DNA interactions were obtained by passing protein solutions over the immobilized oligonucleotides on the surface of the chip and analyzing the resulting sensorgrams.
Specificity of Binding-Initial experiments were designed to check the specificity of binding to uracil-containing DNA and therefore the maximum possible amounts of oligonucleotides ssC and ssU (see "Experimental Procedures") were immobilized on the streptavidin-coated chip (approximately 1100 RU). The mutant D88N and H210N proteins were then passed over both surfaces. As can be seen in Fig. 1 even with these high amounts of immobilized DNA, there was no significant binding to oligonucleotide ssC, demonstrating the absolute requirement for uracil in specific recognition. When the wild-type enzyme was tested, no binding was observed to oligonucleotides ssC or ssU. The most obvious interpretation for this observation would be the rapid removal of uracil by the active enzyme making it impossible to detect a stable enzyme⅐DNA complex. This was confirmed by testing the binding of mutant enzyme before and after injection of the wild-type enzyme. Fig. 3 shows that following application of the active enzyme, the mutant D88N was no longer capable of interacting with the surface, indicating that almost all of the available uracil had been removed. The same result was obtained with mutant H210N (not shown). The much lower binding displayed after removal of the uracil by the wild-type enzyme indicates that neither the wild-type nor the mutants have significant affinity for the abasic sites generated in the single-stranded DNA by the uracil-excision reaction.
Comparison between Single-and Double-stranded Oligonucleotides-Two different nonbiotinylated oligonucleotides (ssG and ssA, see "Experimental Procedures") were used to create double-stranded DNA (dsU/G and dsU/A) using immobilized, biotinylated oligonucleotides ssU, and dsC/G using immobilized, biotinylated ssC. As with ssC, dsC/G showed no indications of binding with the wild-type enzyme nor either of the mutants (results not shown). Fig. 4 shows the relative binding of a single concentration of mutant H210N to four surfaces coated with the same amount (approximately 200 RU of singlestrand oligonucleotides) of ssC, ssU, dsU/G and dsU/A (similar results were obtained with both mutants, data not shown). Both double-strand oligonucleotides bound protein with reduced affinity compared with ssU, and binding to dsU/A was considerably reduced compared with dsU/A. As with the singlestranded uracil-containing oligonucleotides, treatment with wild-type UDG effectively abolished subsequent binding by wild-type or mutant enzymes, indicating that neither the wildtype nor mutant enzymes display significant affinity for the abasic sites in double-stranded DNA produced by the uracilexcision reaction (Fig. 5).
To obtain a quantitative comparison, we took advantage of the fact that equilibrium was reached for all these interactions, and therefore the equilibrium dissociation constant could be determined by injecting a range of concentrations and plotting the response at equilibrium versus the concentration of injected protein. For these assays as well as for kinetic analysis (see below), the level of immobilized oligonucleotides was further reduced to approximately 40 RU to avoid possible artifacts due to mass transport limitations. Furthermore, it has been suggested (20) that the measurement of K D values by equilibrium binding assays is affected by the amount of "receptor" used, and considerable deviations can be observed from the true values if the levels are too high. Whereas the overall response was small, the data obtained in this way fitted very well to an equation describing a simple bi-molecular interaction obeying the law of mass action (see "Experimental Procedures"). The results of these experiments for the mutant H210N are summarized in Table I. An example of the fits obtained using Equation 3 is shown in Fig. 6 for binding to the ssU oligonucleotides. This figure also demonstrates that there is no substantial difference between the two mutant enzymes, D88N and H210N, and similar good fits were obtained for the other oligonucleotides (Table I).
Estimation of Kinetic Constants-To analyze the interaction with the uracil-containing oligonucleotides further, the kinetic constants were determined. An example is shown in Fig. 7 for the binding of the H210N mutant to the dsU/G oligonucleo-tides. Fig. 7 shows the fit of the association rate (A) and that of the dissociation rate (B) obtained using Equations 1 and 2 (see "Experimental Procedures"), which describe a homogeneous single-site interaction. Data were obtained with several different concentrations of protein and for the three different oligonucleotides and are summarized in Table I. In all cases the fits were statistically highly significant, and there was no evidence of deviations due to mass transport effects, although these were observed when higher amounts of oligonucleotides were immobilized (not shown). The interaction with the ssU oligonucleotides is characterized by the fastest association rate and the slowest dissociation rate. The difference in affinity between the two double-strand oligonucleotides is mainly due to an over 10-fold faster association rate for dsU/G compared with dsU/A. From the kinetic constants, the equilibrium dissociation constant could also be independently estimated from the equation K D ϭ k d /k a . As can be seen in Table I there is very good agreement between the K D values calculated in this way and those determined directly by the equilibrium binding experiments described above, confirming the validity of the fits. DISCUSSION Structural studies show unambiguously that recognition of uracil by UDGs requires insertion of the uracil base into a pocket in the enzyme, which for dsDNA can only be achieved by FIG. 3. Uracil excision by the wild-type enzyme abolishes UDG binding to ssDNA. Immobilized ssU oligonucleotide was allowed to interact with D88N before and after injection of an equal concentration of wild-type enzyme. The arrows point to the start and end of each injection. The sharp decreases in the signal after the interaction are due to the regeneration step to remove bound protein (see "Experimental Procedures"). The RU values represent the magnitude of the response compared with the baseline before the first injection. An exact doubling of the response was observed after injection of the second oligonucleotide. Additional injections did not increase the response any further. Marked points indicate the following: 1) beginning of injection of biotinylated single-strand oligonucleotides; 2) end of injection; 3) beginning of injection of second single-strand oligonucleotides; and 4) end of injection (a high concentration of oligonucleotides was used, and because the stock was dissolved in water, dilution of the buffer occurred resulting in the sudden "jump" of the response at point 4. This is not so obvious at point 3 because of the fast binding of the oligonucleotides).
"flipping" the deoxyuridine nucleotide into an extrahelical conformation (4 -6). However, these structural results cannot explain the mechanism of this recognition process. Two questions in particular need to be answered. First, does the enzyme locate uracils by scanning the DNA in a "one-dimensional" diffusion process (22) or by simple bimolecular collision; and second, does the enzyme actively promote the flipping of the uracil from a stacked conformation, or does it recognize spontaneously flipped bases?
The affinity of native and mutant UDG, for single-stranded or double-stranded uracil-free DNA, is too low to be measured using the BIAcore system. This indicates that the residence time of the enzyme on ordinary DNA is very short and makes it unlikely that the enzyme is able to scan along the DNA for any appreciable length. That the rates of association and dissociation from uracil-DNA are very well modeled by single processes (see Equations 1 and 2 and Fig. 7) rather than by the biphasic model required if scanning were in operation (i.e. E ϩ DNA(U) 3 E:DNA(U) 3 E:U(DNA)) further suggests that direct binding of uracil in the DNA is the only process taking place.
Whereas our observations appear to rule out facilitated diffusion as an option for locating uracil in DNA, substrate location might be made more efficient by electrostatic steering of the positively charged DNA-binding face of UDG (4, 6) toward the negatively charged DNA molecule during bimolecular collision events. Such an oriented "hopping" process (22,23) would certainly serve to reduce the effective dimensionality of the search in a similar, albeit more crude, manner than that achieved by one-dimensional diffusion. This would also make sense of data suggesting that UDG interacts with DNA in a "distributive" manner (24), but shows effects that would tally with a scan length of 1.5-2 kilobases prior to dissociation from DNA (25). A hopping mechanism is also supported by the structural data (6), which suggests that UDG would have to dissociate from DNA after a uracil-excision event to allow the free uracil product to be released, a requirement that would be difficult to reconcile with a sliding mechanism.
Introduction of a single uracil base into the single-and double-stranded oligonucleotides produced a dramatic increase in the affinity of the catalytically inactive mutants for the single-and double-stranded DNA over that observed for uracilfree DNA. The relative affinity for uracil-DNA was strongly dependent on the structural context of the uracil, in the order ssU-DNA Ͼ dsU/G-DNA Ͼ Ͼ dsU/A-DNA, for both mutants. As neither the native nor the mutant enzymes showed any significant affinity for the abasic site that results from the uracilexcision reaction (Figs. 2 and 5), this increased affinity can be attributed to the interactions between the enzyme and the destacked uracil base, which are essentially identical in all three systems. This order of relative affinity can be understood in terms of the work required to destack the uracil and make it available to the binding pocket; being least in ssDNA where no base pair is disrupted, more for the "wobble" G:U base pair, and most for the fully stacked Watson-Crick A:U base pair. Affinity for uracil in DNA rather than for any features of DNA per se is consistent with the ability of UDG to act on ssDNA as well as dsDNA, more or less irrespective of sequence context. The differences in affinity for the DNA molecules between the two mutants are very small, confirming the suggestion that both of these residues have purely catalytic roles in the enzyme mechanism (4,6).
Neither the native nor the mutant enzymes showed any apparent affinity for abasic sites, and binding of the active wild-type enzyme to uracil-DNA ligands was not detectable. Association with uracil-DNA and its subsequent hydrolysis by the wild-type enzyme, though undetected, must have taken place as demonstrated by the abolition of subsequent binding of the catalytically inactive mutants. The lack of a detectable enzyme⅐DNA complex during catalysis is consistent with the observed fast on-rate and low affinity for the abasic reaction product and suggests that the actual rate of bond hydrolysis is not limiting.
Two broad classes of base-flipping enzyme can be defined; sequence specific DNA-modifying enzymes, and sequence-independent DNA-modifying enzymes. A sequence-specific enzyme, such as a restriction methyltransferase, interacts with an extrahelical base within a defined sequence context that can be recognized in a fully stacked conformation. Once bound to its cognate DNA sequence, a sequence-specific enzyme can simply wait for the thermal breathing motion of the DNA to flip its target base spontaneously (9,12). In contrast, a sequenceindependent enzyme, such as uracil-DNA glycosylase, must recognize the presence of its target base directly. Unlike some lesions recognized by DNA repair enzymes, which cause significant distortion in the structure of the double helix, uracil is a potentially difficult target to detect. In a G:U mismatch, the wobble of the uracil into the major groove might enable recognition in situ of the presence of uracil, however, a U:A base pair, which is also efficiently repaired, is a fully stacked Watson-Crick base pair with no protrusions. The pattern of functional groups presented by an A:U base pair in the major groove is different from the patterns presented by either of the normal G:C or A:T base pairs and might therefore provide a means for in situ recognition. However, the major groove pattern for A:U is also very different from that presented by G:U, and it is difficult to imagine how an enzyme would achieve simultaneous specific major (or minor) groove recognition for these very different base pairs in dsDNA while also recognizing unpaired uracil in ssDNA. A much simpler model, consistent with the relative strengths of binding we observe for uracil in these different contexts, would suggest that UDG is recognizing "flipped out" uracil. These findings are supported by the rates for association and dissociation of either mutant from the different oligonucleotides. The association rates are markedly different in the order ssU-DNA Ͼ dsU/G-DNA Ͼ Ͼ dsU/A-DNA suggesting that the enzyme⅐U-DNA complex formation is dependent on the relative strength of any base pairing. The dissociation rates are relatively similar and suggest that once the complex is formed, the enzyme dissociation is from an essentially identical complex in all cases.
The limited interaction between UDG and DNA-containing bases other than uracil is a function of the exquisitely selective specificity pocket of the enzyme, which will only allow uracil and some close analogues to enter and bind (4 -6). The displacement of several bound water molecules from the pocket upon binding of uracil is entropically favorable, and the formation of three hydrogen bond contacts to uracil in the active site (4,6), as well as the insertion of a hydrophobic side-chain into the space left in the duplex by the flipped nucleotide (6), is a stable arrangement. Further stabilization will be provided by the DNA-binding channel, which makes favorable contacts with the distorted backbone geometry generated by a flipped nucleotide (6). This suggests that molecular impact of the enzyme onto the DNA and thermal motion of DNA, together with the stabilization of the helix distortion provided by the DNAbinding channel of the enzyme, would be sufficient to drive the complete process of recognition, binding, and catalysis without penalty.
The question of whether UDGs are indeed facilitating inherent base flipping in the duplex, or just binding to opportunistically flipped-out bases cannot be demonstrated solely from our results and the available structural information. However, this could be addressed by measuring energy changes on interaction using a technique such as microcalorimetry. This approach is currently being investigated for the mutant proteins used in this study.