Báo cáo khoa học: A truncated form of DNA topoisomerase IIb associates with the mtDNA genome in mammalian mitochondria doc

The relaxation activity is inhibited by etoposide and other inhibitors of eucaryotic type II enzymes.The DNA topo-isomerase II copurifies with mitochondria and directly associates with mt

A truncated form of DNA topoisomerase IIb associates

with the mtDNA genome in mammalian mitochondria

Robert L Low1, Shayla Orton1and David B Friedman2

1

Department of Pathology and2Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, CO, USA

Despite the likely requirement for a DNA topoisomerase II

activity during synthesis of mitochondrial DNA in

mam-mals, this activity has been very difficult to identify

convin-cingly.The only DNA topoisomerase II activity conclusively

demonstrated to be mitochondrial in origin is that of a type

II activity found associated with the mitochondrial,

kineto-plast DNA network in trypanosomatid protozoa [Melendy,

T., Sheline, C., and Ray, D.S (1988) Cell 55, 1083–1088;

Shapiro, T.A., Klein, V.A., and Englund, P.A (1989) J Biol

Chem 264, 4173–4178].In the present study, we report the

discovery of a type DNA topoisomerase II activity in bovine

mitochondria.Identified among mtDNA replicative

pro-teins recovered from complexes of mtDNA and protein, the

DNA topoisomerase relaxes a negatively, supercoiled DNA

template in vitro, in a reaction that requires Mg2+and ATP

The relaxation activity is inhibited by etoposide and other

inhibitors of eucaryotic type II enzymes.The DNA topo-isomerase II copurifies with mitochondria and directly associates with mtDNA, as indicated by sensitivity of some mtDNA circles in the isolated complex of mtDNA and protein to cleavage by etoposide.The purified activity can be assigned to a
150-kDa protein, which is recognized by a polyclonal antibody made against the trypanosomal mito-chondrial topo II enzyme.Mass spectrometry performed on peptides prepared from the
150-kDa protein demonstrate that this bovine mitochondrial activity is a truncated version

of DNA topoisomerase IIb, one of two DNA topoisomerase

II activities known to exist in mammalian nuclei

Keywords: mitochondrial DNA topoisomerase; mito-chondrial DNA; mtDNA replication; type II DNA topo-isomerase

Mitochondria in mammalian cells contain multiple copies

of a small (
16 kb) circular duplex DNA genome

(mtDNA) that is produced within mitochondria through

repeated cycles of DNA synthesis [1].The mtDNA genome

encodes 13 polypeptides, each of which is an essential

component of one of the enzyme complexes of the

respiratory chain [2].Consequently, all of the enzymes and

DNA binding proteins required for the replication of

mtDNA are encoded on nuclear chromosomes, and

imported into the organelle.Despite progress made in

characterizing the mtDNA replicative polymerase (DNA

pol c) [3–6], efforts to isolate and study some other

components of the DNA replicative complex responsible

for mtDNA synthesis has proved to be exceedingly difficult

This continues to limit our ability to understand the

biochemistry of how mtDNA replication is carried out

This problem is due both to the low abundance of mtDNA

replicative enzymes in tissues, and to the presence of potent

nuclease activity, and other, ill-defined inhibitors in protein

extracts of mitochondria that block mtDNA replication assays in vitro.Furthermore, the presence of small frag-ments of nuclear DNA in standard preparations of mitochondria has also raised concerns that DNA replica-tion activities attributed to mitochondria could in fact represent nuclear contaminants

One class of enzyme activity likely essential for the successful synthesis of mtDNA is DNA topoisomerase Widely distributed throughout nature, DNA topoiso-merases promote the passage of DNA strands through one another, and relieve the torsional stress in DNA produced for example, during progression of the DNA replication fork, during transcription, and when newly replicated DNA genomes need to disentangled from one another [7].Different types of DNA topoisomerase activity have been identified in prokaryotes and in nuclei

of eucaryotes.The type I and III activities are each ATP-independent and break single DNA strands during catalysis [7].They alter the number of times the two strands of DNA revolve around one another (the linking number), in steps of one.In contrast, the type II activities require ATP, produce double-strand breaks during cata-lysis, and change the DNA linking number in steps of two [8,9].Mammalian nuclei contain two different type II activities, named DNA topoisomerase IIa and IIb, which are encoded by separate genes ([10,11]).The type II enzymes are inhibited by novobiocin, and by a variety of useful anticancer drugs including adriamycin, 4¢-(9-acrid-inylamine)methanesulfon-m-anisidide (m-AMSA), etopo-side (VP-16), and ellipticine [12]

Correspondence to R.L.Low, Department of Pathology B-216,

University of Colorado Health Sciences Center, 4200 East Ninth

Avenue, Denver, CO 80262, USA.

Fax: +1 303 315–6721, Tel.: +1 303 3158024,

E-mail: Robert.Low@UCHSC.edu

Abbreviations: BSA, bovine serum albumin; m-AMSA,

4¢-(9-acridi-nylamine)methanesulfon-m-anisidide.

(Received 15 April 2003, revised 29 August 2003,

accepted 2 September 2003)

In contrast to the nuclear DNA topoisomerases, the

DNA topoisomerase activities present in mitochondria

have been very difficult to purify and identify, especially in

vertebrates.Recently, human mitochondria have been

shown to possess a specific DNA topoisomerase I.This

mitochondrially targeted DNA topo I activity (TOP1mt)

is encoded by a unique gene on chromosome 8q24.3 [13]

The mitochondrial enzyme is highly homologous to

nuclear topo I, and based on its size and properties, no

doubt corresponds to the well-known nuclear-like topo I

activity that had previously been reported in mitochondria

of a variety of cell types [14–19].Recently, human

mitochondria have also been shown to import nuclear

DNA topoisomerase IIIa activity [20].This results from

the use of an alternate transcription start site in the

topo IIIa gene that incorporates a mitochondrial targeting

sequence onto the N-terminus of the protein.Much less is

known about mitochondrial type II enzymes.Thus far,

the only type II DNA topoisomerase conclusively

dem-onstrated to be mitochondrial in origin is an enzyme

denoted DNA topoIImt found within the mitochondrion

of protozoa Crithidia fasciculate and Trypanosoma brucei

[21,22].This enzyme likely plays a role in mtDNA (or

kinetoplast DNA) replication and

catenating/decatenat-ing DNA circles from the kinetoplast network [22].The

topoIImt has been localized at the periphery of the

kinetoplast network by immunohistochemistry [23], and

epipodophyllotoxins and related drugs that promote

cleavage of DNA by topoIImt and other type II enzymes,

have been shown to promote cleavage of kinetoplast

DNA [24,25].Recently, suppression of the trypanosome

topoIImt by RNAi has been shown to cause loss of

kinetoplast DNA [26].In addition to trypanosomes, there

is also evidence for a topoIImt in Dictyostelium discoideum

[27] and Plasmodium falciparum [28].In mammalian

mitochondria, potent endonuclease and topoImt activities

have made it exceedingly hard to detect any type II

activity.Several years ago, a putative type II activity,

identified from catenation/decatenation and unknotting

assays, was reported in mitochondria from human

leukemic cells [29] and calf thymus [30], and partially

purified.Unfortunately, neither activity could be purified

to near homogeneity, nor shown to relax a supercoiled

DNA substrate in an ATP-dependent manner.Despite the

difficulties even finding DNA topoisomerase II in

mam-malian mitochondria, such an activity has been suspected

to be responsible for the cleavage of mtDNA seen in

ciprofloxacin-treated cells [31], and for producing a

common deletion seen in human mtDNA, which

accu-mulates with aging.In the case of this deletion, nucleotide

sequences where the mtDNA is deleted seem to resemble

a nucleotide consensus sequence often targeted by

verte-brate type II DNA topoisomerase activities [32,33]

In the present study, we report that a eucaryotic-like type

II DNA topoisomerase activity is associated with bovine

mtDNA.This activity was recovered from insoluble

com-plexes of mtDNA and mtDNA replicative factors that were

gently isolated from disrupted heart mitochondria.An

analysis of tryptic peptides prepared from the purified

enzyme using mass spectrometry indicates that this

topoIImt activity is a truncated form of DNA

topoiso-merase IIb

Materials and methods

Antibodies Polyclonal antibodies prepared against the trypanosome topoIImt, human DNA topoisomerase IIa, and human DNA topoisomerase IIb were generous gifts of D.Ray, Mol Biol Inst., UCLA, Los Angeles, CA, USA; J Holden, Dept

of Pathology, Utah Health Sciences Center, Salt Lake City,

UT, USA; C.Austin, School of Cell and Mol.Biosci., University of Newcastle upon Tyne, UK, respectively

Isolation of the complex of mitochondrial DNA and its associated proteins

All procedures were carried out at 0–4C, unless otherwise stated.Mitochondria were isolated from fresh ventricular muscle of adult bovine-heart obtained from a local meat processing plant (Hyclone, Greeley, CO, USA).The mito-chondria were recovered from the disrupted heart tissue essentially as described [34], except that minced tissue was ruptured by shearing for 30 s at the lowest (not the highest) speed setting in the Waring blender.To isolate the mtDNA– protein complex, each 40 mL aliquot of mitochondrial suspension was diluted with 140 mL of 30 mM Tris/HCl (pH 8), 4 mM EDTA, 100 mM NaCl, 20 mM potassium glutamate, 10% (w/v) glycerol (buffer A), and gently disrupted with the addition of 0.5% (w/v) Triton X-100 After 30 min, the lysate was centrifuged at 145 000 g for

60 min in a Ti50.2 rotor (Beckman) The supernatant fraction was discarded, and the pellets were pooled and re-suspended in 35 mL of buffer A, without Triton X-100, by repeated Dounce homogenization.After 60 min, this sus-pension was centrifuged at 3000 g for 10 min in a JA20 rotor The loose, tan pellet was discarded and the supernatant was carefully decanted.The supernatant was similarly clarified once more.The final supernatant was then centrifuged

145 000 g for 60 min in a Ti50.2 rotor For experiments requiring intact mtDNA–protein complexes, the dark brown pellet (containing complexes of mtDNA and protein) was re-suspended in 10 mL of Buffer A using a Dounce homogenizer, and stored at 3C.When replication proteins were recovered, the dark brown
mtDNA–protein pellet was re-suspended in 20 mL of 300 mM Tris/HCl (pH 8.8),

900 mM NaCl, 20 mM EDTA, 10 mM dithiothreitol by Dounce homogenization.After > 2 h, the suspension was centrifuged 175 000 g for 60 min in a Ti80 rotor, and the supernatant fraction containing soluble mtDNA replication factors (fraction II) was recovered and stored at 3C

Relaxation and catenation assays for DNA topisomerase II activity

Each relaxation reaction contains in 40 lL: 50 mM Tris/ HCl (pH 7.9), 125 mM NaCl, 7.5 mM Mg(OAc)2, 0.25 mgÆmL)1bovine serum albumin (BSA), 5 mM dithio-threitol, 1.5 mM ATP, 500 ng of pUC19 DNA, and 0.5–4 lL of the fraction being assayed.Relaxation reactions are incubated 60 min at 37C, unless otherwise indicated, and stopped by the addition of 1% (w/v) sodium dodecyl sulfate (SDS).The terminated reactions are then applied to

a 150-mL 0.8% (w/v) agarose-gel cast and run in 40 m

Tris/acetate, 1 mMEDTA (pH 8).After electrophoresis at

1.5 VÆcm)1 overnight, the gel is stained in 0.5 lgÆmL)1

of ethidium bromide and photographed under UV

illumination.One unit of DNA topoisomerase II relaxation

activity is defined as the amount of enzyme that relaxes 50%

(500 ng) of the input supercoiled DNA template in 60 min

Activity is estimated from visual inspection of the gel or by

scanning densitometry

Each catenation reaction contains in 40 lL: 40 mMTris/

HCl (pH 7 9), 125 mM NaCl, 7.5 mM Mg(OAc)2,

0.25 mgÆmL)1 of BSA, 5 mM dithiothreitol, none or

1.5 mMATP as indicated, 500 ng of topologically relaxed

pUC19 DNA, and added enzyme.Reactions are run 30 min

at 37C, and stopped by the addition of 1% SDS, 20 mM

EDTA, and 400 mMNaCl.After heating terminated

reac-tions to 85C for 10 min, reactions are applied to a 150-mL

0.8% agarose gel Electrophoresis and photography of the

ethidium stained gel is carried out as described above

Purification of the mitochondrial DNA topoisomerase II

All steps were carried out at 4C.All buffers contained

0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium

metabisulfite, 0.5 lgÆmL)1leupeptin, and 0.01 mM

pepsta-tin, unless otherwise stated.mtDNA–protein complexes

were isolated from an 80-mL suspension of purified bovine

heart mitochondria, and the soluble proteins subsequently

released from the mtDNA–protein complexes at 900 mM

NaCl were collected, as described above (fraction II, 40 mL;

44 mg total protein).Three milliliters of DEAE-Sepharose

resin was added to the fraction II protein concentrate.After

diluting the suspension fourfold in 5 mMdithiothreitol and

gently mixing for 10 min, the DEAE-Sepharose was

collected at 12 100 g for 10 min in a JA20 rotor (Beckman),

and the supernatant, containing DNA topoisomerase II

activity, was carefully decanted and saved (fraction III,

155 mL; 36 mg protein).Fraction III was applied to a

20-mL (12· 1.7 cm2) hydroxylapatite column equilibrated

in 30 mMTris/HCl (pH 7.9), 20 mMpotassium glutamate,

5 mM dithiothreitol, and 20% (w/v) glycerol (buffer B)

Activity was eluted with a linear 200 mL gradient of

0–1.2Mpotassium phosphate (pH 8.0) in buffer B Active

fractions of DNA topoisomerase II activity eluted near

300 mM potassium phosphate, just prior to those of the

mitochondrial DNA topoisomerase I activity, and were

pooled (fraction IV, 8 mL; 4 mg of protein).Fraction IV

was dialyzed against 1 L of buffer B for 2.5 h, and applied

to a 1.5-mL (2 cm· 0.75 cm2) heparin agarose column

Activity was eluted using a linear 15 mL gradient of 0–1M

NaCl in buffer B.Active fractions of DNA topoisomerase II

activity eluted near 500 mMNaCl and were pooled (fraction

V, 1.3 mL; 0.2 mg of protein) Fraction V was diluted with

6.5 mL of 10 mM dithiothreitol and applied to a 1-mL

column of native DNA-cellulose equilibrated with buffer B

Once loaded, the column was washed with 10 of buffer B,

and eluted with a 10-mL linear gradient of 0–1.1MNaCl

DNA topoisomerase II activity eluted near 350 mMNaCl

Active fractions were pooled (fraction VI, 0.6 mL; 0.024 mg

of protein) and concentrated in a Centricon 30 filter.The

fraction VI concentrate (
60 lL) was diluted threefold

with 10 mMdithiothreitol, and layered onto a linear 4.2 mL

gradient of 15–42% (w/v) glycerol containing 30 m Tris/

HCl (pH 7 9), 0 1 mM EDTA, 10 mM Mg(OAc)2, 5 mM dithiothreitol, 0.1% N-octylglucopyranoside, 1 M NaCl Sedimentation was carried out at 299 000 g in an SW60 rotor (Beckman) for 20 h.Twenty-four fractions were collected dropwise from the bottom of the tube.Peak fractions of activity were saved (fraction VII, 0.3 mL;

2 lg of protein, as estimated from intensity of bands observed on silver-stained SDS/PAGE gels relative to that

of marker proteins).The specific activity of the fraction VII enzyme is
1.7 · 105UÆmg)1

Western blot analysis

A purified enzyme fraction containing about 400 ng of total protein was resolved by electrophoresis on a 7.5% reducing SDS/PAGE gel at 100 V in a Bio-Rad mini-PROTEAN II apparatus with a Tris/glycine buffer system [35].Proteins were transferred by electrophoresis to Immobilon-P mem-brane (Millipore) in the Mini Trans-Blot Cell (Bio-Rad) for

180 vH in 30 mM Tris/HCl (pH 8.3), 0.02% (w/v) SDS, 0.014% (w/v) glycine, 20% (v/v) methanol Blots were briefly stained with 0.2% (w/v) Ponceau S to confirm efficient protein transfer.The blots were blocked with 3%(w/v) BSA in phosphate-buffered saline (NaCl/Pi), 0.5% (w/v) Tween 20 for 1 h at ambient temperature.Incubation with the primary antibody (diluted 1 : 500–1 : 25 000 in blocking buffer) was carried out overnight at 4 C.Sub-sequently, blots were washed extensively with frequent changes of NaCl/Picontaining 0.5% (w/v) Tween 20, at ambient temperature for 2 h.The secondary antibody, which was an anti-rabbit, peroxidase-labeled antibody, was then applied, at a dilution of 1 : 1000.After final washes in NaCl/Pifor 2 h at ambient temperature, protein-immune complexes were visualized by chemiluminescence, according

to the procedure recommended by the manufacturer (ECL, Amersham Life Science)

Protein identification by mass spectrometry Proteins were separated by 1D SDS/PAGE and stained with a low-fixation silver stain [36].Protein bands were individually excised and the silver was removed.Gel slices were equilibrated in 100 mM NH4HCO3 and dehydrated with acetonitrile and vacuum centrifugation.Dehydrated gel slices were then rehydrated with 15 lL 25 mM

NH4HCO3 containing 0.01 lgÆlL)1 modified trypsin (Promega), and trypsin digestion was carried out for > 3 h

at 30C.Peptides were extracted with 60% acetonitrile, 0.1% trifluoroacetic acid, dried by vacuum centrifugation, and reconstituted in 8 lL 0.1% trifluoroacetic acid Pep-tides were then desalted and concentrated into 2 lL 60% acetonitrile, 0.1% trifluoroacetic acid using ZipTipC18 pipette tips (Millipore).0.2 lL was applied to a MALDI target and overlayed with 0.2 lL a-cyano-4-hydroxycin-namic acid matrix.MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO mass spectrometer (Applied Biosystems) operated in reflectron mode.Ions [M + H] corresponding to peptide masses were entered into the MS-FIT database search algorithm (http:// prospector.ucsf.edu/) and the SWISS-PROT, NCBInr and pdbEST databases were searched, allowing for complete carbamidomethylation of cysteine and partial oxidation of

methionine Peptide mass errors of up to 50 p.p.m were

considered during the search

Isolation of the mtDNA–protein complex from mitoplasts

Mitoplasts were prepared from bovine heart

mitochon-dria, essentially as described [37] unless otherwise stated

Briefly, a 20 mL suspension of bovine-heart mitochondria

(25 mgÆmL)1protein) was supplemented with 0.5 mgÆmL)1

bovine serum albumin and 0.1% (w/v) digitonin After gently

stirring the suspension for 15 min at 0C, the mitochondria

were diluted with 75-mL of 5 mM Hepes (pH 8),

0.5 mgÆmL)1 bovine serum albumin, 70 mM sucrose,

220 mMmannitol (buffer C) and disrupted using a 40-mL

Dounce homogenizer (four to-and-fro passes with the
tight

pestle).The homogenate was then centrifuged at 15 000 g for

10 min in a JA-14 rotor (Beckman).The soft, brown

inner-membrane (mitoplast) pellet was collected, and resuspended

in 100-mL buffer C using Dounce homogenization, and

centrifuged 15 000 g for 10 min as before.The washed

mitoplast pellet was resuspended with 10 mL of buffer B, and

Triton X-100 was added to 0.5% After 20 min at 0C, the

mtDNA–protein complex was collected at 30 000 g for

30 min, and resuspended in 1 mL of buffer B minus glycerol

Isolation of the mtDNA–protein complex from

mitochondria sequentially treated with DNase I and

proteinase K

A 20-mL sample of a freshly prepared suspension of

bovine-heart mitochondria was diluted to 220 mL with

30 mMTrisÆHCl (pH 7 7), 50 mMsodium glutamate, 10%

(w/v) sucrose (buffer D), and the mitochondria collected at

15 300 g for 15 min in a JA14 rotor (Beckman).The

brown mitochondrial pellet was resuspended in 25 mL of

buffer D by Dounce homogenization and 4 mMMg(OAc)2

and 0.2 mgÆmL)1pancreatic DNase I were added.DNase

digestion was carried out for 30 min at ambient

tempera-ture, and terminated by the addition of 10 mMEDTA.The

DNase I-treated mitochondria were diluted to 220 mL

with buffer D plus 5 mM EDTA, and then collected at

15 300 g for 15 min in a JA14 rotor.This step is intended

to facilitate removal of DNA fragment debris and residual

DNase The wash step was repeated twice more The final

pellet of mitochondria was resuspended in 200 mL of

5 mMMops (pH 7.4), 5 mMKH2PO4, 1 mMEDTA, 0.3M

sucrose, 0.1% BSA (buffer E), then collected at 15 300 g for

15 min in a JA14 rotor.The mitochondria were resuspended

in 20 mL of buffer E, and 40 mL of 10 mMHepes (pH 7.4),

0.6Mmannitol containing 45 lgÆmL)1of proteinase K was

added, as otherwise modified [20].Protein digestion was

carried out 30 min at 0C, and stopped by the addition of

4 mMphenylmethylsulfonyl fluoride.After 10 min at 0C,

the mitochondria were collected at 12 100 g for 15 min in a

JA20 rotor.The mitochondrial pellet was resuspended in

220 mL of buffer E plus 0.1 mM phenylmethylsulfonyl

fluoride, recentrifuged at 15 300 g for 15 min in a JA14

rotor, and the washed pellet resuspended in 220 mL of buffer

E plus 0.1 mMphenylmethylsulfonyl fluoride.This wash step

was repeated four times.The final pellet of DNase I/

proteinase K treated mitochondria was resuspended in

90 mL of buffer A and the mitochondria were disrupted with

the addition of 0.5% Triton X-100 Complexes of mtDNA and protein were isolated as described above

Results

Recovery of a DNA topoisomerases II activity from isolated complexes of mtDNA and protein When preparations of purified bovine-heart mitochondria are disrupted with the addition of 0.5% (w/v) Triton X-100, the mtDNA and its associated proteins are found to reside

in an insoluble complex, which can be recovered from the

mt lysate through a series of differential centrifugation steps These low and high-speed centrifugation steps eliminate fragments of nuclear DNA–protein complexes that invari-ably contaminate the mtDNA–protein complex.As well, they separate the insoluble complex of mtDNA–protein from > 95% of the mitochondrial protein, which is soluble Subsequent treatment of the isolated complex of mtDNA and protein with 900 mM NaCl releases a fraction of mtDNA replicative proteins and DNA binding proteins from the mtDNA.These proteins (now soluble) are recovered in the supernatant following centrifugation of the high salt extract at 30 000 g for 30 min.In contrast, the mtDNA, which nearly all remains insoluble, is still pelleted During purification of DNA polymerase c released from the mtDNA using successive steps of hydroxylapatite and native DNA cellulose chromatography, and glycerol gradi-ent velocity sedimgradi-entation, we idgradi-entified a eucaryotic type ATP-dependent DNA topoisomerase activity.This activity partially copurifies with the DNA pol c activity.The topoisomerase activity is evident from its relaxation of a negatively supercoiled plasmid DNA in a fairly nonproces-sive fashion (Fig.1A).The rate of DNA relaxation appears constant for 30 min at 37C and the extent of DNA relaxation proportional to added enzyme is in the range of 5–20 ng of protein.The maximal rate of template relaxation requires 1.5 mM ATP with one-half maximal relaxation occurring at 0.25 ± 0.05 mMATP.As observed with other type II activities, a trace level of DNA relaxation occurs in the absence of ATP, presumably due to ATP copurified with the enzyme.While addition of 1.5 mM dATP can substitute for ATP, neither 1.5 mMCTP, UTP, nor GTP supports activity (Fig.1B) The relaxation activity is inhibited by novobiocin.In assays containing 0.5 mM ATP, levels of novobiocin above 200 lM are completely inhibitory.Additional titration experiments indicate that 50% inhibition occurs at a concentration of about 70 lM The mt topoisomerase II lacks DNA gyrase activity as indicated by the failure of the enzyme to supercoil 500 ng of

a relaxed DNA template in standard assays that contained ATP in the range of 0.1–5 mM (data not shown).The fraction V enzyme also lacks detectable DNA ligase activity (< 0.05 UÆlL)1) as assessed using a DNase I nicked plasmid DNA template (data not shown)

The DNA topoisomerases II activity copurifies with mitochondria

To rule out the possibility that the observed DNA topoisomerase II activity was a contaminant, we assessed whether this activity copurified with mitochondria collected

through two successive linear 0.5–2Msucrose gradients for

2 h each at 22 000 r.p.m in an SW28 rotor (Materials and

methods).As expected, the mitochondria recovered from

peak fractions of the second gradient appear relatively free

of nuclear DNA contamination that could provide a source

of topoisomerase activity.This can be seen from the prominent 7.3-, 4.8-, and 4.3-kb EcoRI restriction fragments

of bovine mtDNA that are seen when DNA extracted from

Fig 1 Identification of an ATP(dATP)-dependent mitochondrialDNA topoisomerase and demonstration that the activity cosediments with mito-chondria Photographs of agarose-gel assays are shown.Relaxed and supercoiled (sc) forms of the plasmid DNA are as labeled.(A) Time course Standard agarose-gel, relaxation assays contained 20 ng of fraction VI enzyme, without or with 1.5 m M ATP, and were run 5, 10, 15, or 30 min, as indicated.(B) Activity requires ATP(dATP).Standard relaxation assays were carried out either with none, or 1.5 m M ATP, GTP, CTP, UTP, or dATP, as indicated.Reactions were run for 30 min at 37 C.(C) ATP-dependent DNA topoisomerase II cosediments with mitochondria.Two 12-mL samples of fresh bovine heart mitochondria (40 mgÆmL)1protein) were sedimented through 30 mL, linear 0.5–2 M sucrose gradients (prepared in 30 m M Tris/HCl (pH 8), 75 m M NaCl).Sedimentation was for 2 h at 70 000 g in an SW28 rotor (Beckman), run at 3 C.The visible band of mitochondria from each gradient was removed laterally from the tube using a 16-gauge needle.The mitochondria were pooled and diluted

to 250 mL in 30 m M Tris/HCl (pH 8), 75 m M NaCl (buffer C).The mitochondria were collected at 11 000 r.p.m.for 20 min in a JA14 rotor, resuspended in 10 mL of buffer C and layered onto a second, 30 mL linear 0.5–2 M sucrose gradient which was centrifuged for 2 h at 70 000 g, as described above.Following centrifugation, 15–2 3 mL fractions were collected dropwise from the bottom of the tube.The visible band of mitochondria eluted in fractions 8 and 9.DNA was phenol extracted from a 0.5-mL aliquot of fractions 5 through 14 that were supplemented with 1% SDS.After a treatment with 0.1 mgÆmL)1of RNase A for 40 min at 37 C, each DNA sample was digested with 20 U of EcoR1 for 5 h at

37 C, analyzed by agarose-gel electrophoresis on a 0.8% agarose gel.A photograph of the ethidium-stained gel is shown in (C).The mitochondria

in the remainder of fractions 8 and 9 were pooled, diluted to 32 mL with buffer A and disrupted in the presence of 0.5% Triton X-100 After 30 min

at 3 C, the mtDNA–protein complex was collected by centrifugation at 175 000 g in a Ti80 rotor (Beckman) for 30 min.Proteins released from the isolated mtDNA–protein complex at 900 m M NaCl were then prepared, concentrated to 0.2 mL using a Centricon-10 filter The concentrate was then layered onto a 4-mL, linear 15–42% (v/v) glycerol gradient.Sedimentation was carried out for 20 h at 299 000 g in a SW60 rotor (Beckman) Fractions were collected dropwise from the bottom of the tube and assayed for type II DNA topoisomerase activity.Standard, agarose-gel relaxation assays performed on even numbered fractions between 2 and 12, without and with 1.5 m M ATP are shown in (D).These reactions contained 2 lL aliquots of each fraction assayed and were carried out 30 min at 37 C.

samples of the mitochondria are digested by EcoRI and

analyzed by agarose-gel electrophoresis (Fig.1C).To assess

whether the purified mitochondria still contain

topoiso-merase II activity, the remainder of the mitochondria were

disrupted with 0.5% (w/v) Triton X-100, complexes of the

mtDNA–protein were collected, and a concentrate of

proteins released from the mtDNA at 900 mM NaCl was

prepared and sedimented through a linear glycerol gradient

This velocity sedimentation step has proved to be quite

effective in separating the topoisomerase II activity from the

mitochondrial topoisomerase I and endonuclease G

acti-vities, which both strongly inhibit type II topo assays.As

seen in Fig.1D, ATP-dependent topoisomerase activity is

evident in the gradient, in those fractions collected near the

bottom (fractions 5 and 6), where the peak of DNA

polymerase c activity is also found (data not shown).Thus it

is unlikely that the topoisomerase II activity is a nuclear or

cytoplasmic contaminant

The purified mitochondrial DNA topoisomerase II

activity is sensitive to known inhibitors of eucaryotic type

II enzymes and can catalyze catenation of plasmid DNA

circles.As determined using the fraction V enzyme, DNA

relaxation activity occurs over a fairly narrow range of

Mg2+, NaCl, and pH with maximal rates occurring at

7.5 mMMg2+, 100 mMNaCl, and pH 8.0–8.5, respectively

Relaxation activity shows a strict requirement for Mg2+,

but not Ca2+or Mn2+, in the range of 0.1–15 mMsupports

activity.In addition to novobiocin, etoposide and m-AMSA

(inhibitors of eucaryotic type II enzymes), inhibit the

mitochondrial DNA topoisomerase II activity (Table 1)

Concentrations of each substance that cause 50% inhibition

of the relaxation activity of the fraction V enzyme are listed

In addition to being able to relax negatively supercoiled

DNA templates in the presence of ATP, the mitochondrial

DNA topoisomerase II activity can also promote catenation

of topologically relaxed plasmid DNA circles in the presence

of a DNA crowding agent.As shown in Fig.2, this

catenation activity requires ATP, as expected, and produces

huge networks of interlocked DNA circles that fail to enter

the agarose gel during electrophoresis

The mitochondrial DNA topoisomerases II activity

appears to be associated with mtDNA

An association of DNA topoisomerase II with mtDNA has

been demonstrated by showing that treatment of the

isolated mtDNA–protein complex with etoposide in the presence of SDS promoted cleavage of mtDNA circles into full-length linear mtDNA [38].In this experiment, small samples of a suspension of the isolated mtDNA–protein complex were incubated with or without etoposide in the presence of 1% SDS for 15 min at 37C.After the addition

of proteinase K, the mtDNAs were phenol purified, resolved by agarose-gel electrophoresis and transferred to nitrocellulose paper.The extent of drug mediated cleavage

of the mtDNA was then assessed by Southern blot analysis using a [32P]BamH1-Hpa1 restriction fragment of bovine mtDNA as probe.As seen in Fig.3, treatment with etoposide, in the range of 50–500 lM, converted some mtDNA circles to full-length linear DNA.However, so far,

we have not seen more than about 15% of the input circles linearized (as indicated from scanning densitometry, data not shown), even if the incubation periods at 37 and 64C were extended.Maximal conversion occurs at about 100 lM

of drug.This cleavage reaction required 10 mMMg2+but not the addition of ATP.No DNA cleavage was observed using drug vehicle (dimethylsulfoxide) alone

The purified mitochondrial type II activity can be assigned to a polypeptide of
150 kDa

In order to further identify the mitochondrial DNA topoisomerase II, the enzyme recovered from the isolated mtDNA–protein complex was purified using successive steps of hydroxylapatite, native DNA-cellulose, and heparin agarose chromatography, followed by glycerol gradient

Table 1 Known inhibitors of eucaryotic type II DNA topoisomerases

inhibit the bovine mitochondrialDNA topoisomerases II activity.

Standard ATP-dependent relaxation assays were carried out with the

fraction VI enzyme as detailed in Materials and methods.Novobiocin

assays contained 0.25 m M ATP N-ethylmaleimide assays performed

without added dithiothreitol.

Inhibitors

Concentration that gives 50%

inhibition of relaxation activity 1.Etoposide 70 l M

2 m-AMSA 3 l M

3.Novobiocin 70 l M

4 N-Ethylmaleimide 60 l M

5.Ethidium bromide 0.6 lgÆmL)1

Fig 2 The purified mitochondrialDNA topoisomerase promotes ATP-dependent catenation of relaxed plasmid DNA circles Catenation assays were performed for 30 min at 37 C as detailed in Materials and methods Assays contained 0.5, 1.0, or 2.0 lL of the glycerol gradient pool of mitochondrial DNA topoisomerase II (fraction VII), 5 U of purified mitochondrial DNA topoisomerase I activity, and none or 1.5 m M ATP, as indicated.A photograph of the ethidium stained agarose gel is shown; C, control DNA minus enzyme; rc, relaxed cir-cular form DNA; sc, supercoiled form DNA.

velocity sedimentation.When the fractions of the glycerol

gradient spanning the peak of DNA topoisomerase II

activity were analyzed using a silver-stained SDS/PAGE, a

band
doublet of
150 kDa, within the peak of highest

activity (fraction 6), correlated with the DNA

topoiso-merase II activity (Fig.4A,B).Western blot analysis using

antibodies against trypanosome topoIImt, and human

nuclear topoIIa and topoIIb enzymes provided further

evidence that this 150 kDa polypeptide corresponds to a

DNA topoisomerase II activity (Fig.4C).As shown, the

bovine mitochondrial
150 kDa polypeptide was

recog-nized by antibody prepared against the purified

trypano-somal DNA topoIImt (left most gel, Fig.4C), however, not

by a rabbit nonimmune serum (not shown).As seen in gel

panel 2, this bovine
150 kDa band was also recognized by

antibody made against human nuclear DNA topoIIa, albeit

less well than that seen with the antigen control (panel 3).In

contrast, as evident in panel four, it failed to be recognized

at all by the antinuclear topoIIb antibody.Positive control

blots for antinuclear topoIIa and topoIIb antibodies with human topoIIa and topoIIb antigens are shown in gel panels three and five, respectively

Identification of the mitochondrial type II activity

as a truncated form of DNA topoisomerases IIb Mass spectrometry was used to confirm that the mito-chondrial proteins representing the 150 kDa bands were topoisomerases.Proteins in the activity peak from a glycerol gradient were separated by SDS/PAGE, silver stained and digested in-gel with trypsin protease as described in Materials and methods.Peptide masses acquired by mat-rix-assisted laser desorption/ionization, time of flight mass spectrometry (MALDI-TOF MS) were used in database search algorithms and led to an unambiguous match to human topoisomerase IIb (Fig.5).The bovine homologue was not present in the databases searched.However, a bovine cDNA was found that contained 100% identity with the human sequence (Fig.5)

Human topoisomerase IIb, as well as several other mammalian homologues, has a predicted molecular weight

182 kDa, well above the
150 kDa mobility observed for the mitochondrial proteins.Furthermore, peptide cov-erage was not found past residue 1250 in the human sequence (out of a total of 1621 or 1626, depending on the splice variant), despite nearly all ions being accounted for in the mass spectrum (Fig.5).These findings are consistent with the hypothesis that the mitochondrial enzyme identi-fied from the gel slice is truncated.Although the spectra contain peptides unique to topo IIb, no peptides unique to topo IIa have been encountered.This finding suggests that the mitochondria topo II activity is likely a form of topo IIb, although the spectra do not exclude the possibility that a fragment of topo IIa could also be present

Further evidence that the truncated topo IIb is mitochondrial in origin and not a nuclear contaminant

As the mitochondrial DNA topoisomerase II activity corresponds to a truncated form of the DNA topoisomerase IIb found in nuclei, we decided it was imperative to re-evaluate whether the mitochondrial activity could simply

be a nuclear contaminant.Had the topoisomerase II recovered from the purified mitochondria originated from fragments of nuclear DNA that adhere to mitochondria? Two additional experiments carried out indicate that this is not likely.In the first experiment, samples of mitochondria were treated with 0.1% digitonin to strip away outer membranes and remove nuclear DNA debris that could be adherent to mitochondria.The resultant mitoplasts were then collected, disrupted with 0.5% Triton X-100, and the complex of mtDNA–protein recovered by differential centrifugation.Proteins released from this mtDNA complex

at 600 mMNaCl were then fractionated by glycerol gradient velocity sedimentation and assayed for DNA topoisomerase

II activity.As shown in Fig.6A, agarose-gel electrophoresis

of the mtDNA purified from the mitoplast mtDNA–protein complex and digested with EcoR1 reveals the prominent 7.3-, 4.8-, and 4.3-kb bands characteristic of bovine mtDNA.As seen, the mitoplast preparation appears essentially free of nuclear DNA contaminants.In spite of

Fig 3 Etoposide promotes cleavage of some mtDNA circles of the

mtDNA–protein complex [38] Ten-microliter samples of the

suspen-sion of mtDNA–protein complex were each diluted into 150 lL of

30 m M Tris/HCl (pH 7.9), 125 m M NaCl, 2 m M dithiothreitol, 7.5 m M

Mg(OAc) 2 , 1 5 m M ATP, without or with 50, 100, 250, or 500 l M

etoposide, at 3 C SDS was immediately added to 1% (w/v), and the

reactions were incubated 30 min at 37 C.Proteinase K was then

supplemented to 0.1 mgÆmL)1, and the reactions were further

incu-bated 20 min at 64 C.DNAs were phenol and chloroform extracted

once, ethanol precipitated, and the precipitates collected at 31 000 g

for 30 min in a JA20 rotor.Each precipitated DNA sample was

re-suspended in 40 lL of 40 m M Tris/acetate, 1 m M EDTA (pH 8)

(TAE), plus 1% SDS, and applied to a 150-mL 0.8% agarose gel that

was then run at 1.5 VÆcm)1overnight in a TAE buffer system.DNAs

in the gel were blotted onto nitrocellulose membrane by capillary

transfer in 1.5 M NaCl, 0.15 M sodium citrate (pH 7) (10 · NaCl/Cit),

as described [69].Hybridization was carried out overnight at 68 C in

6 · NaCl/Cit, 0.25% (w/v) nonfat dried milk, with a heat-denatured

[5¢- 32

P]BamH1-Hpa1 restriction fragment of the D-loop region

sequence of bovine mtDNA (
10 8 dpmÆlg)1), as probe.After

extensive washing of the filter, as detailed [69], autoradiography was

carried out for 1 h at )80 C using Kodak XR5 film.A photograph of

the autoradiogram is shown.The full-length linear form of bovine

mtDNA was identified using a Hpa1-digested sample of purified

bovine mtDNA, as shown.

this, the topoisomerase assays performed on the glycerol

gradient reveal a vigorous topoisomerase II activity,

peak-ing in fractions 8 and 9.This indicates strongly that there is

DNA topoisomerse II activity associated with mtDNA as previous experiments suggested.Furthermore, as shown in Fig.6B, Western blot analysis of the topoisomerase II peak

Fig 4 The mitochondrialDNA topoisomerase II activity is associated with a » 150-kDa polypeptide that is recognized by an anti-trypanosome topoIImt Ig (A) Silver-stained SDS/PAGE gel of the active, mitochondrial DNA topoisomerase II fraction of the glycerol gradient velocity sedimentation purification step.A 200-lL sample of the fraction V enzyme (200 l) was layered onto a 4-mL linear 15–42% (v/v) glycerol gradient containing 30 m M Tris/HCl (pH 7.9), 300 m M NaCl, 10 m M Mg(OAc) 2 , 5 m M dithiothreitol, 0.05% (w/v) n-octylglucoside.Sedimentation was carried out at 299 000 g in a SW60 rotor (Beckman) for 20 h at 3 C.Twenty, five-drop fractions were collected from the bottom of the tube Proteins in a 100-lL aliquot of fractions 4, 6, and 8 were each precipitated in 10% (w/v) trichloroacetic acid, the protein precipitants collected at

31 000 g for 30 min in a JA20 rotor, were resuspended in 20 lL of 50 m M Tris/HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 0.7 M

2-mercaptoethanol, 0.05% (w/v) bromophenol blue Samples were heat-denatured 3 min at 94 C, and run through a 7.5% reducing, SDS/PAGE gel, at 100 V in a Tris/glycine buffer system [34].A photograph of the gel stained with silver [34], is shown in (A).Marker, molecular size standards: myosin (200 kDa), b-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa).(B) Standard agarose-gel relaxation assays of glycerol gradient contained in 40 lL: a 2-lL aliquot of fraction 4, 6, or 8, without or with 1.5 m M ATP, as indicated.Assays were carried out for 60 min at 37 C.A photograph of the agarose gel is shown.(C) Western analysis using antibodies against trypanosomal topoIImt, and human nuclear DNA topoisomerases II a and b reveals cross reactivity with the bovine mitochondrial enzyme.Blots were probed with diluted antibodies and were developed by chemiluminescence, and exposed to X-ray film.Primary antibodies were used at the following dilutions: anti-trypanosome (1 : 500); anti-topoIIa (1 : 500) and anti-topoIIb (1 : 25 000).The blot prepared with the fraction V of the bovine mitochondrial DNA topoisomerase II (400 ng) was probed with either the anti-trypanosome Ig gel panel 1), the anti-topoIIa Ig (SDS-gel panel 2), or the anti-topoIIb Ig (SDS-(SDS-gel panel 4), respectively, as shown.The anti-topoIIa and topoIIb Igs were tested with a positive control of either recombinant human topoIIa (
150 ng) or topoIIb antigen (
200 ng) provided by the supplier of the antibody (SDS-gel panels 3 and 5, respectively).Relative sizes of prestained standards run in an adjacent lane are shown at the left of the gel.

of the glycerol gradient reveals a faint, immunoreactive

band of
150 kDa, in agreement with size of the purified

activity.This result further suggests that the apparent

truncation of the topoisomerase IIb does not simply result

from in vitro proteolysis during the several-step enzyme

purification.In contrast to the mtDNA–protein complexes

recovered from the mitoplasts, the complexes of

nuc-lear protein and DNA fragments removed from the mitochondria by digitonin fail to yield any detectable DNA topoisomerase II activity, when proteins bound to this DNA are released by high salt, fractionated by glycerol gradient velocity sedimentation, and assayed (Fig.6C)

In a second experiment, a 20-mL sample of bovine-heart mitochondria was treated successively with DNase I and

Fig 5 Tryptic peptides from bovine topoisomerases IIb identified by mass spectrometry Protein bands were excised after 1D SDS/PAGE and digested in-gel with trypsin protease as described in Materials and methods.(A) Silver-stained SDS/PAGE gel separating proteins present in fraction 6 from the second glycerol gradient.Arrows indicate the 150 kDa proteins that were individually subjected to in-gel digestion with trypsin protease as described in Materials and methods.(B) Matrix-assisted laser desorption/ionization, time of flight (MALDI-TOF) mass spectrum of tryptic peptides isolated from the lower 150 kDa protein band.Asterisked ions (m/z ¼ 1070.48, 1115.64, 1122.59, 1128.64, 1131.57, 1159.58, 1264.60, 1270.65, 1296.66, 1315.70, 1331.66, 1357.74, 1436.68, 1461.78, 1500.80, 1640.85, 1830.98, and 2423.19 Da – left to right) match predicted tryptic peptide masses (plus one amu) from human topoisomerase IIb (MOWSE Score ¼ 1.35 · 10 9

) Ions labeled T match expected trypsin autolytic peptides (m/z ¼ 842.50, 1045.56 and 2211.09 Da) and were used to internally calibrate the mass spectrum to a mass accuracy of within

50 p.p.m Ions labeled K match background peptides derived from keratin that were also present in controls x-axis, mass-to-charge ratio (m/z); y-axis, relative ion intensity.(C) Amino acid sequence of human topoisomerases IIb.Residues contained within the predicted tryptic peptides matched by the MALDI-TOP MS data are indicated in boldface.The only bovine sequence found to significantly match the MALDI-TOF MS data was the cDNA 211850 MARC 2BOV, which encodes a peptide containing a 100% match to the human amino acid sequence (shaded in gray, amino acids 725–904).

proteinase K to degrade any DNA topoisomerase II that

could be adherent to mitochondria.After several cycles of

washing to remove proteolytic debris and any trace

proteinase K activity, the mitochondria were disrupted

with the addition of 0.5% Triton X-100, and the mtDNA–protein complexes recovered, and proteins released from the mtDNA fractionated by glycerol gradient velocity sedimentation.DNA topoisomerase II activity, measured either by ATP-dependent catenation or relaxation assays, was identified in glycerol gradient fractions 7, 8, and

9, as expected.The amount of activity recovered was about 75% that obtained in the mitoplast experiment (see Fig.6)

In contrast, no topoisomerase II activity could be recovered

if the mitochondria were first disrupted with 0.5% Triton X-100 prior to the addition of the proteinase K

Discussion

In this study, we present biochemical evidence that mam-malian mitochondria contain a catalytically active, trun-cated form of DNA topoisomerase IIb.This activity copurifies with mitochondria collected over successive sucrose gradients, and the activity is associated with purified complexes of mtDNA and protein that are recovered from isolated mitochondria and digitonin-treated mitoplasts using steps that eliminate nuclear DNA contaminants Unlike the well-characterized nuclear form of DNA topo-isomerase IIb that consists of a 180-kDa polypeptide and relaxes DNA in a processive fashion in vitro [39,40], the polypeptide of the mitochondrial DNA topoisomerase IIb is

150 kDa in size, and its relaxation activity acts fairly nonprocessively.The mitochondrial form of DNA topo-isomerase IIb retains sensitivity to well-known, clinically useful inhibitors of DNA topoisomerase II activity, but the enzyme fails to be recognized by a topoisomerase IIb-specific antibody prepared against C-terminal epitopes not present in DNA topoisomerase a.Furthermore, mass spectrometric analysis of the mitochondrial polypeptide shows an absence of peptides predicted from the C-terminal sequence.These findings suggest that the mitochondrial activity lacks the
30-kDa C-terminal domain of the nuclear enzyme

This C-terminal truncation does not appear to be an

in vitroproteolytic artifact, as several other polypeptides identified in these fractions of mitochondrial protein, including the polypeptides for DNA polymerase c and its accessory factor, adenylate kinase, apoptosis inducing factor, and endonuclease G, each has a size that is what should be expected (R.Low, K.Fang and D.Friedman, unpublished data).Furthermore, we have been able to detect this
150-kDa polypeptide (as shown in Fig.6), but not any protein band of
180-kDa on a Western blot at an early step in purification suggesting that this form of the enzyme is not a proteolytic artifact but what resides in mtDNA–protein complexes

Proteolytic degradation of DNA topoisomerase II during its purification can be a major problem with some types of tissue.For example, purification of DNA topoisomerase II isoenzyme forms from calf thymus that is notoriously rich in protease activity typically yields active 120- and 140-kDa fragment artifacts of DNA topoisomerase II activity [41,42] However, with heart tissue and isolated heart mitochondria used in this study, proteolysis during enzyme isolation appears much less problematic.In contrast to thymic and other types of cells, adult myocytes contain relatively few lysosomes [43], a major source of proteolysis.Furthermore,

Fig 6 The truncated topo IIb arises from mtDNA not from

contami-nants of nuclear DNA fragments (A) Identification of DNA

topoisomerase II activity among proteins released from mitoplast

mtDNA–protein complexes and fractionated by glycerol gradient

velocity sedimentation.Proteins released at 600 m M NaCl from

mtDNA–protein complexes of mitoplasts (see Materials and methods)

were concentrated in a Centricon 10 filter (Amicon).This concentrate

(150 mL) was then run through a 15–42% glycerol gradient as

des-cribed in the Fig.4 legend, except that the glycerol gradient contained

1 M , not 300 m M NaCl.Topo II assays plus and minus ATP on active

fractions were carried out, are shown.An EcoR1 digest of DNA

phenol-purified from a sample of the mtDNA–protein complex (25%

of total) is also shown.(B) SDS/PAGE and Western blot analysis of

glycerol gradient fractions 8 plus 9.One-half of this pool was used for

each gel analysis.See Fig.4 legend for details.(C) Topo II assays,

minus and plus ATP, performed on glycerol gradient fractionation of

proteins released at 600 m M NaCl from insoluble
outer membrane

complexes of nuclear DNA–protein that were released from mitoplasts

with digitonin, and recovered at 16 000 g for 20 min.Velocity

sedi-mentation was carried out as described in the legend of Fig.4.

Agarose-gel analysis of an EcoR1 digest of 4 lg nuclear (nuc) DNA

recovered from the
outer membrane fraction is shown.Relative

positions of mtDNA EcoR1 fragments in a far lane (not seen) are

indicated.