SuzukiOndrovicová, Eva Kutejová and Carolyn K. Tong Liu, Bin Lu, Irene Lee, Gabriela  by Nucleotide and Protein SubstrateMitochondrial Lon Protease Is Regulated DNA and RNA Binding by theEnzyme Catalysis and Regulation:

doi: 10.1074/jbc.M309642200 originally published online January 22, 20042004, 279:13902-13910.J. Biol. Chem. 

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DNA and RNA Binding by the Mitochondrial Lon Protease IsRegulated by Nucleotide and Protein Substrate*

Received for publication, September 2, 2003, and in revised form, January 21, 2004Published, JBC Papers in Press, January 22, 2004, DOI 10.1074/jbc.M309642200

Tong Liu‡, Bin Lu‡, Irene Lee§, Gabriela Ondrovicova¶, Eva Kutejova¶, and Carolyn K. Suzuki‡�

From the ‡Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine andDentistry of New Jersey, Newark, New Jersey 07103, the §Department of Chemistry, Case Western Reserve University,Cleveland, Ohio 44106, and the ¶Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21,845 51 Bratislava, Slovak Republic

The ATP-dependent Lon protease belongs to a uniquegroup of proteases that bind DNA. Eukaryotic Lon is ahomo-oligomeric ring-shaped complex localized to themitochondrial matrix. In vitro, human Lon binds specif-ically to a single-stranded GT-rich DNA sequence over-lapping the light strand promoter of human mitochon-drial DNA (mtDNA). We demonstrate that Lon binds GT-rich DNA sequences found throughout the heavy strandof mtDNA and that it also interacts specifically withGU-rich RNA. ATP inhibits the binding of Lon to DNA orRNA, whereas the presence of protein substrate in-creases the DNA binding affinity of Lon 3.5-fold. Weshow that nucleotide inhibition and protein substratestimulation coordinately regulate DNA binding. In con-trast to the wild type enzyme, a Lon mutant lacking bothATPase and protease activity binds nucleic acid; how-ever, protein substrate fails to stimulate binding. Theseresults suggest that conformational changes in the Lonholoenzyme induced by nucleotide and protein sub-strate modulate the binding affinity for single-strandedmtDNA and RNA in vivo. Co-immunoprecipitation ex-periments show that Lon interacts with mtDNA polym-erase � and the Twinkle helicase, which are componentsof mitochondrial nucleoids. Taken together, these re-sults suggest that Lon participates directly in the me-tabolism of mtDNA.

The ATP-dependent Lon (La) protease is a multi-functionalenzyme conserved from archaea to mammalian mitochondria(1–6). Mitochondrial Lon is a homo-oligomeric complex inwhich each monomer carries separate sites for the binding andhydrolysis of both ATP and protein substrate (7, 8). Lon selec-tively degrades abnormal polypeptides, thus serving a qualitycontrol function in protein biogenesis (9–12). The energy re-quirement of Lon is mechanistically similar to that describedfor other ATP-dependent proteases such as ClpAP and the

proteasome (13–20). The binding of substrate polypeptide toLon stimulates its ATPase activity (21). ATP hydrolysis isrequired for the processive unfolding of a substrate that per-mits peptide bond cleavage. Conformational changes withinthe Lon holoenzyme likely coincide with the cycle of ATP bind-ing and hydrolysis, ADP dissociation, and ATP rebinding, aswell as with the binding and hydrolysis of protein substrate.

Lon belongs to a unique group of proteases that also bindDNA. Other proteases or protease components that bind DNAinclude the human adenovirus proteinase (AVP),1 the adipo-cyte-enhancer binding protein 1 (AEBP1), Gal6p/bleomycin hy-drolase, and the 19 S particle of the 26 S proteasome. AVPrequires two co-factors for maximal protease activity: viralDNA and a viral peptide produced by AVP proteolysis. It isproposed that AVP utilizes its nonspecific DNA binding activityto locate its viral protein substrates (22). By contrast, theAEBP1 repressor binds specifically to the adipocyte enhancer 1element. The carboxypeptidase activity of AEBP1 is stimulatedby adipocyte enhancer 1 element DNA and is required fortranscriptional repression; however, the protein substrates hy-drolyzed by AEBP1 remain unknown (23, 24). In yeast, Gal6phas been shown to negatively regulate galactose metabolismand also to hydrolyze bleomycin, which is widely used in cancerchemotherapy. Studies show that DNA-binding mutants ofGal6p fail to protect yeast from bleomycin toxicity (25, 26).Recent work has shown that the 19 S component of the 26 Sproteasome binds specifically to the GAL1–10 promoter andfunctions in nucleotide excision repair and transcriptional elon-gation in yeast (27–29). Lon shares structural and functionalsimilarity with the ATPase subunits of the 19 S particle withintheir respective AAA domains. Based on these findings, it ispredicted that the DNA binding activity of Lon is important forits physiological function.

In vivo studies show that Lon has a role in DNA maintenanceand expression. In Saccharomyces cerevisiae, strains lackingmitochondrial Lon suffer large deletions in mtDNA and do notprocess and express mitochondrial RNA transcripts that con-tain introns (30–32). In Caulobacter crescentus, Lon is respon-sible for the cell cycle-dependent turnover of the CcrM DNAmethyltransferase; lon mutants fail to initiate DNA replicationand to complete cell division (33). In Escherichia coli, datasuggest that Lon degrades the protein(s) that determine thehelical density of F-type DNA mini-plasmids that replicateindependently of the bacterial chromosome (34). In addition,

* This work was supported by National Institutes of Health GrantGM61095, Basil O’Connor Scholars Award-March of Dimes GrantFY99-797, and a grant from the Foundation of University of Medicineand Dentistry of New Jersey (to C. K. S.); by National Institutes ofHealth Grant GM067172 (to I. L.); by Slovak Grant Agency GrantVEGA 2/2086/22 (to E. K.); and by a grant from the Twinning Program-National Science Foundation/National Academy of Sciences (to C. K. S.,I. L., and E. K.). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

� To whom correspondence should be addressed: New Jersey MedicalSchool, University of Medicine and Dentistry of New Jersey, Dept. ofBiochemistry and Molecular Biology, 185 S. Orange Ave., Newark, NJ07103. Tel.: 973-972-1555; Fax: 973-972-5594; E-mail: suzukick@umdnj.edu.

1 The abbreviations used are: AVP, human adenovirus proteinase;AEBP1, adipocyte-enhancer binding protein 1; BSA, bovine serum al-bumin; EMSA, electrophoretic mobility shift assay; FITC, fluoresceinisothiocyanate; mtDNA, mitochondrial DNA; MOPS, 4-morpholinepro-panesulfonic acid; NTA, nitrilotriacetic acid; AMP-PCP, adenosine 5�-(�,�-methylenetriphosphate); POLG, polymerase �.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 14, Issue of April 2, pp. 13902–13910, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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studies performed in various bacterial species suggest that Lonregulates the protein levels of transcription factors that controldevelopment, antibiotic production, or pathogenesis (35–40).

In vitro studies from the 1980s examined the relationshipbetween the DNA binding and enzymatic activities of E. coliLon. A. Markovitz and co-workers (41, 42) purified the lon(capR9) gene product as a DNA-binding protein; independ-ently, Chung and Goldberg demonstrated that this protein wasidentical to the ATP-dependent Lon/La protease (43). Bothgroups found that E. coli Lon bound to DNA nonspecifically,and both showed that the ATPase activity of Lon was stimu-lated by single- and double-stranded DNA. More recent in vitrostudies have demonstrated that bacterial, mouse, and humanLon bind specifically to GT-rich DNA sequences (44–46). Al-though the sequence specificity of DNA binding is evolutionar-ily conserved, experiments show that mammalian Lon bindssingle-stranded DNA, whereas bacterial Lon binds double-stranded DNA (44–46). The binding site for human Lon invitro is contained within a GT-rich DNA oligonucleotide whosesequence overlaps the light strand promoter of human mtDNA.

Little is known about the sequence specificity and regulationof nucleic acid binding by Lon and whether Lon interacts withproteins involved in mtDNA metabolism. In this study, wedemonstrate that Lon binds not only to GT-rich DNA sequencesfound throughout the guanine-rich heavy strand of humanmtDNA but also to GU-rich RNA. ATP inhibits Lon binding toDNA and RNA, whereas protein substrate increases the affin-ity of the protease for nucleic acid. We propose that DNA andRNA binding by the human mitochondrial Lon protease isregulated by conformational changes within the holoenzymethat are induced differentially by nucleotide and protein sub-strate. In cultured cells human Lon associates with compo-nents of mitochondrial nucleoids, which are large multi-proteincomplexes bound to mtDNA that support the stability, expres-sion, and inheritance of the mitochondrial genome. We showthat mtDNA polymerase � and the Twinkle helicase co-immu-noprecipitate with Lon. These results are consistent with thenotion that Lon participates directly in the metabolism ofmtDNA.

EXPERIMENTAL PROCEDURES

Expression of Recombinant Human Lon—A cDNA encoding humanLon carrying an amino-terminal hexahistidine tag followed by a linkerregion (MGHHHHHHDYDIPTTENLYFQGAHM) fused to methionine115 was expressed in the E. coli Rosetta strain (Novagen). Bacteriawere grown at 37 °C in 3% bactotryptone, 2% yeast extract, 1% MOPS,pH 7.2, 100 �g/ml ampicillin, 34 �g/ml chloramphenicol and inducedwith 1 mM isopropyl-�-D-thiogalactopyranoside for 90 min at 37 °C.Relatively low levels of human Lon protein were expressed recombi-nantly as high levels of expression lead to an increase in Lon degrada-tion and a decrease in cell viability. From �7 g of bacteria, 1–2 mg ofpure Lon was obtained after the three-step purification describedbelow.

Purification of Recombinant Human Lon—The cells were harvestedand suspended in Solution A (0.2 M NaCl, 25 mM Tris buffer, pH 7.5,20% glycerol, 2 mM �-mercaptoethanol) and sonicated on ice. The lysatewas centrifuged, and the supernatant was loaded onto a P11 column(Whatman; phosphocellulose) equilibrated with Solution A. The columnwas washed with Solution A and eluted with a linear NaCl gradient(0.2–2.0 M NaCl, 25 mM Tris, pH 7.5, 20% glycerol, 2 mM �-mercapto-ethanol). The fractions eluted at 0.62–0.75 M NaCl were positive for the�100-kDa Lon monomer as demonstrated by immunoblotting withanti-Lon antiserum. These fractions were pooled, and the protein wasconcentrated, subjected to size exclusion chromatography using aSephacryl S-300-HR column (Sigma), and then eluted using Solution A.The Lon-containing fractions were collected and loaded onto a Ni2�-NTA column (Qiagen). The column was washed three times with NTAbuffer (0.5 M NaCl, 25 mM Tris, pH 7.5, 20% glycerol) and once withNTA buffer containing 50 �M imidazole. Lon bound to the column waseluted in three steps with NTA buffer containing 0.1, 0.2, or 0.3 M

imidazole. Purified human Lon was analyzed by SDS-PAGE and Coo-

massie Brilliant Blue staining and by immunoblotting using an anti-Lon antiserum.

In Vitro Mutagenesis of Human Lon cDNA—The cDNA encoding theLon S855A mutant in which serine at position 855 was replaced byalanine was produced using QuikChange XL site-directed mutagenesis(Stratagene). Complementary oligonucleotide primers (coding sequence5�-acccccaaggacggcccagccgcaggctgcaccatcgtc) were used to introduce asingle point mutation that was verified by DNA sequencing. LonS855Ais a fusion protein that carries the same amino-terminal hexahistidinetag and linker as described above.

Glutaraldehyde Cross-linking—Cross-linking experiments were car-ried out as previously described (7, 8). SDS-denatured or native Lon(300 pmol of monomer) was cross-linked using 0.1% glutaraldehyde forthe time periods as indicated and then visualized on a 3% SDS-PAGEstained with Coomassie Brilliant Blue.

� DNA Cleavage, Protease, and ATPase Assays—Lon (5 pmol ofmonomer) was incubated with �DNA (0.4 �g) in a 40-�l reaction con-taining 2 mM NaCl, 2 mM Tris, 1 mM MgCl2, 0.5 mM CaCl2 for the timeperiods as indicated at 37 °C; the reactions were run on a 0.9% agarosegel in TBE buffer. ATP-dependent degradation of casein was measuredas described previously (46, 47). Briefly, wild type Lon (7.4 pmol ofmonomer) or LonS855A (8.4 pmol of monomer) was incubated with 160pmol of FITC-casein in a 40-�l reaction containing 50 mM Tris-Cl, pH8.1, 150 mM NaCl, 2 mM dithiothreitol, 10 mM Mg(OAC)2 in the presenceor absence of 2 mM ATP at 37 °C. At various time points, the aliquotswere withdrawn and quenched with 60% trichloroacetic acid and BSAand centrifuged at 4 °C. The level of fluorescence in the supernatantrepresenting acid-soluble peptides derived from FITC-casein was meas-ured spectrophotometrically. A calibration curve using known concen-trations of FITC-casein was used to calculate moles of FITC-peptidesgenerated. The ATPase activity of Lon was determined by measuringthe hydrolysis of [�-32P]ATP to [�-32P]ADP using thin layer chromatog-raphy (48) and phosphorimaging quantification. Purified Lon (12 pmolof monomer) or LonS885A (13 pmol of monomer) was incubated in a30-�l reaction containing 50 mM Tris-Cl, pH 8.1, 2 mM Mg(OAC)2, 150mM NaCl, 2 mM ATP, and 50 nM [�-32P]ATP at 37 °C. The aliquots werewithdrawn at various time points and quenched with 0.5 M formic acid.Casein-stimulated ATPase activity was assayed in the same manner,except 440 pmol of casein was included in the reaction mixture.

Oligonucleotides Used in Nucleic Acid Binding Assays—The follow-ing oligonucleotides were assayed: single-stranded oligonucleotideLSPas (5�-aat aat gtg tta gtt ggg ggg tga) as previously described (45,46) and single-stranded mtDNA sequences 1as (5�-ggc gta ggt ttg gtc tagggt), 2as (5�-gta gag ggg gtg cta tag ggt), 3as (5�-gta tgg ggg taa tta tggtgg), 4as (5�-gga ggg ggg ttg tta ggg ggt), 5as (5�-cga ggg tgg taa gga tggggg), and 6as (5�-ggg gag ggg tgt tta agg ggt). The positions of oligonu-cleotides 1s–6s within human mtDNA (GenBankTM sequenceNC_001807) are: 7114–7134, 8262–8282, 8395–8415, 10936–10956,12387–12407, and 15526–15546, respectively. The LSPas RNA oligo-nucleotide was 5�-aau aau gug uua guu ggg ggg uga. The respectivecomplementary sequences were also tested. Synthetic DNA or RNAoligonucleotides were end-labeled, unincorporated radioactivity was re-moved, and the probes were adjusted to the same specific activity aspreviously described (46). DNA and RNA oligonucleotides were sus-pended as a stock solution in 10 mM Tris, pH 8.0. The oligonucleotidescorresponding to LSPas DNA and RNA as well as to oligonucleotides G,G1, G2, and G3 form high order complexes resulting from four or morecontiguous guanine residues. The uncomplexed forms of these oligonu-cleotides were gel-purified before end labeling.

Electrophoretic Mobility Shift Assay—EMSA was performed as pre-viously described (46) using radiolabeled probe (3.2 pmol) and purifiedLon (5–10 pmol of monomer) incubated in a 30-�l reaction in thepresence or absence of unlabeled LSPas or LSPs DNA or RNA (200-foldexcess), polydeoxyinosinic-deoxycytidylic acid (poly(dI-dC), 50 ng/reac-tion), or oligonucleotide A (200-fold excess).

Nitrocellulose Filter Binding Assay—DNA binding reactions (30 �l)containing Binding Buffer (20 mM Hepes, pH 7.4, 2 mM MgCl2, 0.02 mM

dithiothreitol), human Lon (5–10 pmol of Lon monomer), and 4 �l oflabeled oligonucleotide (3.2 pmol) were incubated at 37 °C for 30 min.Binding Buffer (200 �l) was added to each reaction, which was appliedimmediately to a nitrocellulose filter pre-equilibrated with bindingbuffer. The nitrocellulose filters were washed with 10 ml of BindingBuffer and analyzed by liquid scintillation counting.

DNA binding affinity of Lon for LSPas was determined using thefilter assay. Total binding was determined by incubating Lon (0.8 pmol)with LSPas probe (8–16,000 fmol) in the presence and absence of casein(4 pmol) for 30 min at 37 °C. Nonspecific binding was determined byadding unlabeled LSPas (800 pmol) to the reactions. Specific binding

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was calculated as total binding minus nonspecific binding. Prism 3.0software was used to calculate the Kd by nonlinear regression as well asthe standard deviation (based on data obtained from three independentexperiments).

Transient Transfection, Co-immunoprecipitation, and Immunoblot-ting—293T cells were transfected as previously described (46) withpCDNA3.1 or with pCDNA3.1 constructs carrying cDNAs encodingeither polymerase � (POLG) or Twinkle (kindly provided by JohannesSpelbrink, Tampere, Finland). Both POLG and Twinkle were taggedwith the Myc epitope at their respective carboxyl termini. Forty-eighthours post-transfection, the cell extracts were prepared, and endoge-nous human Lon was immunoprecipitated using a rabbit anti-Lonantibody bound to protein A-agarose. Immunoprecipitated proteinswere immunoblotted with an anti-Myc epitope 9E10 antibody (SantaCruz, SC-40) or with an affinity-purified anti-Lon antibody using theECL chemiluminescence detection system (46). To determine the spec-ificity of the co-immunoprecipitations, control experiments were per-formed in which the anti-Lon antibody was omitted. In addition, cellextracts (20 �g) were immunoblotted with the anti-Myc antibody todetermine the expression level of transiently expressed POLGand Twinkle.

RESULTS

Purification of Recombinant Human Lon and Analysis of ItsEnzymatic Activities—To analyze the DNA binding and enzy-matic activities of Lon in vitro, the protein was expressed andpurified from E. coli. Expression of a Lon fusion protein carry-ing an amino-terminal hexahistidine tag was induced withisopropyl-�-D-thiogalactopyranoside for 90 min; at this timepoint maximal expression and minimal degradation of Lon wasobserved by immunoblotting (Fig. 1A). Cell extracts were sub-jected to cation exchange chromatography (Fig. 1B, lane 2), andfractions containing Lon were pooled, concentrated, and fur-ther purified by size exclusion chromatography (Fig. 1B, lane3). Lon-containing fractions were collected and finally purifiedby Ni2�-NTA affinity chromatography (Fig. 1B, lane 4). A Lonmutant, LonS855A, in which a conserved serine at position 855at the proteolytic active site was replaced by alanine, was alsoexpressed and purified using the same protocol as for wild typeLon (data not shown). Glutaraldehyde cross-linking experi-ments demonstrated that human Lon was a homo-oligomericcomplex composed of six or seven �100-kDa subunits (Fig. 1C),similar to that shown for yeast Lon (7). Complete cross-linkingof SDS-denatured Lon resulted primarily in monomers migrat-ing at �100 kDa (lane 1). Complete cross-linking of the nativeprotease resulted in a large molecular mass complex running atthe top of the gel (lane 2), and partial cross-linking revealed thatthis complex was composed of six or seven subunits (lane 3).

Because we aimed to study the DNA and RNA binding ac-tivity of Lon, experiments were performed to confirm that thepurified protein was nuclease-free. Fig. 1D shows that after thefinal purification step Lon was free of endonucleases; � DNAremained stable even after a 24-h incubation at 37 °C with Lon(lanes g and h). By contrast, � DNA was completely degradedafter incubation under the same conditions with Lon isolatedafter the size exclusion step (Fig. 1D, lane f). The absence ofother nucleases was shown by the stability of single- and dou-ble-stranded DNA and RNA oligonucleotides incubated withpurified Lon (data not shown).

To demonstrate the protease activity of purified Lon, caseincovalently labeled with fluorescein isothiocyanate (FITC-ca-sein) was used as a substrate (46, 47, 49). The energy-depend-ent degradation of casein has been routinely employed to char-acterize bacterial, yeast, and mammalian Lon (1, 8, 46, 50). Thecleavage of FITC-casein by Lon was measured spectrofluoro-metrically as an increase in acid-soluble fluorescent caseinpeptides. Fig. 2A demonstrates that wild type Lon degradedFITC-casein in the presence of ATP but not in its absence; therate of hydrolysis was 0.48 mol of casein/mol Lon/s. By con-trast, LonS855A failed to degrade FITC-casein either in thepresence or in the absence of ATP.

Substrate-stimulated ATPase activity of wild type Lon andthe LonS855A mutant was determined using [�-32P]ATP andthin layer chromatography (48). Wild type Lon exhibited abasal ATPase activity hydrolyzing 0.81 mol of ATP/mol Lon/s.The addition of casein stimulated hydrolysis 2-fold to 1.89 molof ATP/mol Lon/s. By contrast, LonS855A lacked detectableATPase activity either in the presence or in the absence ofcasein. Taken together these results demonstrate that purifiedwild type Lon is an active ATP-dependent protease and thatLonS855A is an enzymatic mutant that lacks both ATPase andprotease activity.

Sequence-specific DNA and RNA Binding by Human Lon inVitro—Previous work has demonstrated that mouse and hu-man Lon specifically bind to a single-stranded GT-rich DNAoligonucleotide referred to as LSPas (45, 46). The sequence ofLSPas overlaps with the light strand promoter anti-sensestrand of human mtDNA. By contrast, the complementaryLSPs sequence is not bound by Lon. Electrophoretic mobilityshift assays showed that human Lon specifically bound to DNAas well as RNA oligonucleotides carrying the LSPas sequence(Fig. 3, A and B). LSPas DNA binding by Lon led to a shift in

FIG. 1. Expression and purification of human Lon. A, immunoblot of Lon induction by isopropyl-�-D-thiogalactopyranoside (IPTG). B,Coomassie Blue-stained gel of crude cell extract (lane 1); Lon-containing fractions from cation exchange chromatography (lane 2); size exclusionchromatography (lane 3); and Ni2�-NTA affinity chromatography (lane 4). C, complete glutaraldehyde cross-linking (30 min) of SDS-denatured anddialyzed Lon demonstrates monomers of �100 kDa (lane 1). Complete (lane 2) and partial (1 min, lane 3) cross-linking of native Lon revealed thatthe complex is composed of six or seven monomers. The protein was visualized on a 3% SDS-PAGE stained with Coomassie Brilliant Blue. D,cleavage of � DNA by Lon-containing fractions. Shown are � DNA alone (lanes a and e); � DNA � Lon isolated after size exclusion chromatography(lanes b and f); � DNA � Lon eluted with either 0.2 M imidazole (lanes c and g) or 0.3 M imidazole from Ni2�-NTA-agarose (lanes d and h); and 1-kbDNA markers (lane m; New England Biolabs).

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probe mobility that was competed by a 200-fold excess of unla-beled LSPas DNA. No competition was observed upon theaddition of poly(dI-dC) (50 ng) or deoxyribo-oligonucleotide A(200-fold excess) (Fig. 3A). Lon also bound specifically to LSPasRNA and not to its complementary LSPs RNA sequence (Fig.3B). The binding to LSPas RNA was competed by the additionof LSPas DNA (see Fig. 5F). Lon binding to other GU-richRNAs was also observed; this binding was competed by bothLSPas RNA and DNA but not by spermidine (data not shown).Lon failed to bind to the respective complementary sequencesof the GU-rich RNAs tested. These results demonstrate thatthe specificity of nucleic acid binding by Lon is determined bythe sequence of purine and pyrimidine bases and not by thesugar phosphate backbone.

Effect of Monovalent and Divalent Cations on the Interactionof Lon with DNA—To determine the metal dependence of Lonbinding to DNA, EMSA was performed in the presence andabsence of Mg2�, Mn2�, Ca2�, Na�, and K�. The mitochondrialmatrix is a known store of Ca2�, and several matrix enzymesuse Mg2� or Mn2� as a co-factor. Within the matrix peakconcentrations of Ca2� are reportedly �1 mM (51, 52), andconcentrations of Mg2� and Mn2� are at �0.2–1.5 mM and �1mM, respectively (53–55). Fig. 3C shows that Lon bound toLSPas DNA in the absence as well as in the presence of Mg2�,Mn2�, and Ca2� at physiological concentrations. Inhibition ofbinding was observed only at concentrations beyond those doc-umented to occur within the matrix. The concentrations of Na�

and K� in “resting” mitochondria are both reported to be �15mM (56). We found that increasing Na� concentrations hadlittle effect on the binding of LSPas DNA, and K� concentra-tions affected binding only at �100 mM.

Lon Binds to GT-rich DNA Sequences Found throughout theMitochondrial Genome—The human mitochondrial genome is a16,571-base pair closed circular double-stranded DNA. Theheavy strand of mtDNA has a greater mass than the light strandbecause of its high content of guanine residues. Throughout theheavy strand there are clusters of 4–12 guanine residues every�100 bases, and most of these are flanked by at least one thy-midine nucleotide. We therefore tested the binding of Lon to DNAoligonucleotides corresponding to G-rich sites within the heavystrand as well as their respective complementary sequences.Nitrocellulose filter binding assays demonstrated that Lon boundpreferentially to G-rich heavy strand oligonucleotides, referred toas antisense 1–6 (1as–6as) rather than to the complementarysense sequences (1s–6s) (Fig. 4A). Further analysis demon-strated that Lon also bound to a DNA oligonucleotide consistingof only guanine residues (Fig. 4B). Introduction of flanking thy-midine residues on either side of a hexaguanine cluster increased

Lon binding to DNA. Taken together these results demonstratethat Lon binds to G-rich DNA sequences in vitro and suggest thatLon interacts with G-rich sites present throughout the mitochon-drial genome.

ATP Binding Inhibits the Interaction of Lon with DNA andRNA—ATP binding and hydrolysis are essential to the cata-lytic cycle by which Lon degrades protein substrates. The effectof various nucleotides on the binding of LSPas DNA by Lon wastested using the nitrocellulose filter assay. Increasing concen-trations of ATP inhibited DNA binding (Fig. 5A). In the pres-ence of 1, 2, and 5 mM ATP, Lon binding to the LSPas probe was�80, 50, and 30% of untreated samples, respectively. A similarinhibitory effect was observed using increasing concentrationsof ADP, albeit to a lesser extent than ATP. The addition ofnonhydrolyzable analogs of ATP also blocked Lon binding toDNA, demonstrating that ATP hydrolysis was not required forinhibition (Fig. 5A). The inhibitory effects of GTP and GDPwere similar to those observed for ATP and ADP, respectively.Little or no influence on DNA binding by Lon was observedupon the addition of increasing amounts of AMP or GMP (Fig.5A) or dATP, dGTP, dTTP, or dCTP (data not shown). Lonbinding to LSPas RNA was also antagonized by ATP and AMP-PCP and was also competed by the addition of 200-fold excessLSPas DNA (Fig. 5F).

Substrate-stimulated DNA Binding by Lon—To test whetherthe binding of Lon to LSPas DNA was influenced by proteinsubstrate, EMSA was performed in the presence or absence ofeither casein, which is degraded by Lon or native BSA, which isnot degraded (57). The addition of casein substantially in-creased the binding of Lon to the LSPas probe, whereas BSAhad little effect (Fig. 5B). Affinity binding studies demon-strated that the addition of casein increased the affinity of Lonfor LSPas DNA 3.5-fold. Fig. 5C shows that in the absence ofcasein the approximate Kd of Lon binding to LSPas DNA was711.2 � 150.6 nM, whereas in the presence of casein the ap-proximate Kd was 201.1 � 46.5 nM. The slight decrease inspecific binding to LSPas at high concentrations may be causedby factors within the experimental system that we do notunderstand and thus cannot control.

Nucleotide and Protein Substrate Coordinately RegulateDNA Binding by Lon—Once nucleotide inhibits the DNA bind-ing activity of Lon, does protein substrate stimulate DNA bind-ing? Conversely, once protein substrate stimulates DNAbinding, does added nucleotide lead to DNA release? To ad-dress the first question the following order-of-addition experi-ment was performed. In Step 1, AMP-PCP was added to theLon-LSPas reaction for 15 min; in Step 2, casein was added,and the reaction was incubated for an additional 15 min. AMP-

FIG. 2. Enzymatic activities of wild type Lon and LonS855A. A, ATP-dependent proteolysis of the model substrate FITC-casein assayedspectrophotometrically. Lon or LonS855A was incubated with substrate in the presence or absence of 2 mM ATP. B, hydrolysis of ATP was assayedby thin layer chromatography. Lon or Lon S855A was incubated with [�-32P]ATP in the presence and absence of casein.

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FIG. 3. Specific binding of Lon toDNA and RNA oligonucleotides. Bind-ing of Lon to 32P-labeled LSPas DNA andRNA oligonucleotides was analyzed byEMSA. A, LSPas DNA probe was incu-bated either alone (lane 1); � Lon (lane 2);� Lon � 200-fold excess unlabeled LSPas(lane 3); � Lon � 50 ng poly(dI-dC) (lane4); or � Lon � 200-fold excess DNA oligo-nucleotide A (lane 5). B, LSPas or LSPsRNA probe alone (lanes 1 and 4); � Lon(lanes 2 and 5); � Lon � 200-fold excessunlabeled LSPas or LSPs RNA (lanes 3and 6). C, the effect of various cations onLSPas DNA binding was analyzed byEMSA. Lon was incubated with probe inthe presence of the indicated cationconcentrations.

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PCP was used in these experiments because it does not supportthe degradation of casein (data not shown). Fig. 5D shows thatAMP-PCP blocked Lon binding to LSPas DNA in a dose-de-pendent manner and that the subsequent addition of caseinstimulated DNA binding. The level of casein stimulation de-pended on the concentration of AMP-PCP added in the firststep; at the higher concentration of AMP-PCP, a lower stimu-lation of DNA binding was observed. To address whether theincrease in substrate-stimulated DNA binding by Lon is nucle-otide-sensitive, the following order-of-addition experiment wasperformed. In Step 1, casein was added to the Lon-LSPasreaction for 15 min; in Step 2, AMP-PCP was added, and thereaction was incubated for an additional 15 min. Fig. 5D showsthat casein stimulated Lon binding to LSPas DNA and that thesubsequent addition of AMP-PCP led to the release of LSPas.Increasing concentrations of AMP-PCP resulted in increasedLSPas dissociation. These results demonstrate a functionalrelationship between nucleotide inhibition and protein sub-strate stimulation of DNA binding by Lon.

Nucleotide and protein substrate did not influence DNAbinding by the Lon S855A mutant. LonS855A lacks both pro-tease and ATPase activity (Fig. 2); however, it retains DNAbinding activity (Fig. 5E). Although LonS855A bound to LSPasDNA, casein failed to stimulate DNA binding (Fig. 5E), andATP had no effect on this binding (data not shown). EitherLonS855A cannot interact with ATP and casein, or its affinityfor nucleotide and protein substrate is substantially reduced.These results suggest that unlike wild type Lon, LonS855Adoes not undergo the conformational changes required to pro-mote or block DNA binding in vitro.

Although ATP and protein substrate modulate DNA bindingby Lon, we have not found that DNA oligonucleotides influencethe ATPase or protease activities of the enzyme in vitro (datanot shown). ATPase and protease assays were performed as inFig. 2 in the presence and absence of LSPas or LSPs DNAoligonucleotides at �M final concentrations; no changes in en-zyme kinetics were observed. A previous study showed that

DNA stimulated ATP-dependent proteolysis by E. coli Lon (43),whereas another study showed that DNA inhibited this activity(41). It is difficult to compare our results with those of earlierexperiments that used larger double- and single-stranded DNAsequences at different final concentrations.

Lon Associates with Proteins Localized to MitochondrialNucleoids—The binding of Lon to mtDNA sequences in vitroprompted us to investigate whether Lon associates with pro-tein components of mitochondrial nucleoids, which are largemulti-protein complexes that have been inferred to be the unitsof mtDNA replication, transcription, and inheritance (58–61).Recent work has shown that mtDNA POLG as well as theputative mtDNA helicase Twinkle localize to human mitochon-drial nucleoids (62). 293T cells were transfected with plasmidconstructs for the transient expression of either POLG or Twin-kle; both proteins carried a carboxyl-terminal Myc epitope tag.Forty-eight hours post-transfection, endogenous Lon was im-munoprecipitated from protein extracts of detergent solubilizedcells, and the interaction between Lon and POLG or Twinklewas assayed by Western blotting. Fig. 6 demonstrates that bothPOLG and Twinkle co-immunoprecipitated with Lon. Overex-pression of POLG or Twinkle did not alter the level of immu-noprecipitated Lon (Fig. 6) nor the level of Lon in total cellularprotein extracts (data not shown). These results support theinvolvement of Lon in regulating mtDNA metabolism in vivo.

DISCUSSION

Model for the Regulation of Nucleic Acid Binding by Lon inVitro—The results presented here show that purified Lon bindsspecifically to GT-rich DNA and GU-rich RNA and that nucle-otide and protein substrate modulate oligonucleotide bindingby Lon. ATP binding inhibits the interaction of Lon with DNAand RNA, whereas protein substrate stimulates binding. Theartificial substrate casein was used in these studies because ithas been employed routinely to characterize the proteolyticactivity of Lon from many different species. The stimulation ofDNA binding occurs through a labile interaction between the

FIG. 4. Specific binding of Lon to GT-rich sequences found on the heavy strand of human mtDNA and to oligonucleotidesconsisting of only guanine residues. A, Lon binding to DNA oligonucleotides corresponding to various G-rich regions of human mtDNA (see“Experimental Procedures”) assayed by filter assay. B, Lon binding to DNA oligonucleotides consisting only of guanine or guanine and thymidineresidues.

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protease and substrate, because we do not observe a stablesupershifted complex representing Lon, DNA, and casein (Fig.5B). We are not able to assay the effect of casein on RNA binding,because casein contains contaminants that lead to RNA degra-dation. Once DNA binding has been stimulated by substrate, theaddition of ATP or its nonhydrolyzable analogs leads to nucleicacid release. Conversely, once DNA binding is inhibited by nu-cleotide, this inhibition is overcome by the addition of proteinsubstrate resulting in enhanced DNA binding (Fig. 5D).

We propose the following model to explain the enzymatic andDNA binding activities of Lon in vitro (Fig. 7). The Lon com-plexes purified in this study most likely consist of monomers inan ADP-bound form or in an ADP-depleted state (Fig. 7A). NoATP is added during the purification of Lon; thus, all or most ofthe bound ATP is hydrolyzed to ADP by the end of the proce-dure because the enzyme has a weak ATPase activity even inthe absence of substrate (Fig. 2B). Nucleotide-free subunitsadopt a conformation that either exposes an increased numberof DNA and RNA binding sites or converts low affinity sites tohigh affinity sites for nucleic acid binding (Fig. 7A). The inter-action of protein substrate with the Lon complex leads to ADPrelease, resulting in an ADP-depleted form of the enzyme (Fig.

7B). Previous studies with E. coli Lon have shown that proteinsubstrate stimulates the release of ADP (21). Substrate-stim-ulated release of ADP promotes a conformation of the holoen-zyme that increases further the number or affinity of DNA- andRNA-binding sites. The stoichiometry of Lon binding to nucleicacid in the presence or absence of protein substrate, ATP, orADP is not known. In the presence of excess ATP, the majorityof nucleotide-binding sites within the Lon complex are occupiedby ATP, resulting in a conformation with a low affinity for DNAor RNA (Fig. 7C); thus any bound nucleic acid is released. Thepresence of both ATP and protein substrate leads to a cycle ofpeptide bond hydrolysis and ADP release. How Lon-mediatedproteolysis is coordinated with DNA or RNA binding either invivo or in vitro remains to be determined.

The stimulation of the DNA binding activity of Lon by pro-tein substrate suggests that nucleic acid binding is regulatedby specific proteins. We demonstrate that casein stimulatesDNA binding by Lon, whereas BSA does not, consistent withprevious work showing that Lon selectively degrades caseinbut not native BSA (57). Protein substrate binding and hydrol-ysis may regulate a cycle of mtDNA binding and release (Fig.7). Transient association of Lon with GT-rich DNA is supported

FIG. 5. Nucleotide and protein substrate regulate DNA and RNA binding by Lon. A, the relative effect of nucleotides on Lon binding tothe LSPas DNA probe was calculated as a percentage of the specific binding to LSPas in the presence of nucleotide divided by that bound in theabsence of nucleotide. B, effect of casein or BSA on the binding to LSPas DNA by Lon (10 pmol of monomer). �2 pmol of casein or �1.5 pmol ofBSA were used. C, casein increases the affinity of Lon for LSPas DNA. In the absence of casein the Kd is 711.2 � 150.6 nM, whereas in its presencethe Kd is 201.1 � 46.5 nM. The calculations are based on results from three independent experiments. D, order-of-addition experimentsdemonstrating that nucleotide and protein substrate coordinately regulate DNA binding by Lon. Panel a, Step 1 only. Lon (5–10 pmol) wasincubated with LSPas probe in the presence or absence of AMP-PCP (1, 5, or 10 mM) for 15 min at 37 °C. Panel b, Steps 1 and 2. same as panela except that after Step 1, casein (4 pmol) was added followed by an additional 15-min incubation. Panels c and d, in Step 1, Lon (5–10 pmol) wasincubated with probe in the presence of casein (2 or 4 pmol) for 15 min at 37 °C. In Step 2, AMP-PCP (1, 5, or 10 mM) was added to the reactionfollowed by an additional 15-min incubation. The relative effect of AMP-PCP or casein in Steps 1 and 2 was calculated as a percentage of the specificbinding of Lon to LSPas alone. E, casein-stimulated DNA binding requires an intact proteolytic active site. The effect of casein or BSA on bindingto LSPas DNA by the Lon S855A protease mutant is shown (same conditions as in B). F, the relative effect of ATP, AMP-PCP, and LSPas DNAon the binding of Lon to the LSPas RNA probe was calculated as for A.

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by the low affinity of DNA binding (Fig. 5C). However, it is alsopossible that some proteins interact with Lon and activatebinding, even though they are not targets for degradation. Suchproteins would induce a conformational change in the Loncomplex promoting nucleic acid binding but not protein degra-dation. In so doing, another level of regulation could be imposedupon the DNA and RNA binding function of Lon.

Studies have shown that mtDNA is associated with the innermembrane and is bound by large multi-protein complexes re-ferred to as mitochondrial nucleoids. Although the majority ofmitochondrial Lon is soluble in the matrix, a fraction of Lon isassociated with the inner membrane (63). Localization of Lonat the inner membrane may be explained by its interactionwith mtDNA. Because Lon also interacts with mtDNA polym-erase � and the putative mtDNA helicase Twinkle (Fig. 6), Lonitself may be a component of nucleoids where it functions todegrade, process, or influence the activity of POLG, Twinkle, orother nucleoid proteins and thus regulate mtDNA metabolism.

Functional Significance of Lon Binding to GT-rich Sequencesin mtDNA—The heavy strand of human mtDNA exhibits ahigh frequency of 4–12 contiguous guanine residues every�100 bases, many of which are flanked by at least one thymi-dine residue. One can envisage that those G-rich clusters thatfunction as Lon-binding sites are bound transiently by theprotease during mtDNA replication or transcription when theheavy and light strands are dissociated and single-stranded. Ithas been shown that single-stranded DNA and RNA containingstretches of guanine bases have a propensity for forming gua-nine “tetraplexes” or “quadruplexes,” which are four-strandedstructures based on the hydrogen-bonded guanine tetrad orG-quartet (64–66). Experiments are required to determinewhether Lon binds to discrete linear G-rich sequences or toquadruplex-like structures and whether Lon binding affectsintramolecular or intermolecular interactions between theseG-rich sites.

Proteins that regulate mtDNA replication and transcriptionare largely unknown. The DNA and RNA binding activity ofLon provides a mechanism by which to recruit the protease tothe mitochondrial genome where it can degrade or processproteins involved in mtDNA and mtRNA metabolism. In addi-tion, the binding of Lon to G-rich sites found throughoutmtDNA may impart pause sites that function as checkpointsduring replication, transcription, or translation. The binding ofmtDNA by Lon may directly inhibit or promote the processivityor timing of replication and/or transcription. Experimentsaimed at exploring these potential functions and at identifying

proteins complexed with Lon bound to mtDNA are currentlyunderway.

Acknowledgments—We are grateful to D. Fritz and Drs. M. Z.Humayun, M. Modak, C. S. Newlon, and K. Singh for helpful discus-sions and critical reading of this manuscript.

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