DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions

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Contentslistsavailableat SciVerseScienceDirect

Molecular

&

Biochemical

Parasitology

DNA

polymerase

beta

from

Trypanosoma

cruzi

is

involved

in

kinetoplast

DNA

replication

and

repair

of

oxidative

lesions

Bruno

Luiz

Fonseca

Schamber-Reis

a,d

_,

_Sheila

_Nardelli

b

_,

_Carlos

_Gustavo

_Régis-Silva

a

_,

Priscila

Carneiro

Campos

a

_,

_Paula

_Gonc¸

_alves

_Cerqueira

a

_,

_Sabrina

_Almeida

_Lima

a

_,

Glória

Regina

Franco

a

_,

_Andrea

_Mara

_Macedo

a

_,

_Sergio

_Danilo

_Junho

_Pena

a

_,

Christophe

Cazaux

e

_,

_{Jean-Sébastien}

_Hoffmann

e

_,

_Maria

_Cristina

_Machado

_Motta

c

_,

Sergio

Schenkman

b

_,

_Santuza

_Maria

_Ribeiro

_Teixeira

a

_,

_Carlos

_Renato

_Machado

a,∗ a_Department_of_Biochemistry_and_Immunology,_Institute_of_Biological_Sciences,_Federal_University_of_Minas_Gerais,_Belo_Horizonte,_Brazil b_{Microbiology,}_Immunology_and_Parasitology_Department,_Federal_University_of_São_Paulo,_São_Paulo,_Brazil

c_Hertha_Meyer_Cellular_Ultra_structure_Laboratory,_Institute_of_Biophysics_Carlos_Chagas_Filho,_Federal_University_of_Rio_de_Janeiro,_Brazil

d_School_of_Medical_Sciences,_Superior_Learning_and_Development_Center_(CESED),_Campina_Grande,_Paraíba,_Brazil

e_Institute_of_Pharmacology_and_Structural_Biology,_UMR5089_CNRS_and_University_of_Toulouse,_Toulouse,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received19April2011

Receivedinrevisedform9February2012 Accepted14February2012

Available online 24 February 2012

Keywords: DNArepair Trypanosomacruzi Oxidativestress Oxoguanine Kinetoplast

a

b

s

t

r

a

c

t

SpecificDNArepairpathwaysfromTrypanosomacruziarebelievedtoprotectgenomicDNAand

kine-toplastDNA(kDNA)frommutations.Particularpathwaysaresupposedtooperateinordertorepair

nucleotidesoxidizedbyreactiveoxygenspecies(ROS)duringparasiteinfection,being

7,8-dihydro-8-oxoguanine(8oxoG)afrequentandhighlymutagenicbasealteration.Ifunrepaired,8oxoGcanleadto

cytotoxicbasetransversionsduringDNAreplication.Inmammals,DNApolymerasebeta(Pol␤)ismainly

involvedinbaseexcisionrepair(BER)ofoxidativedamage.HoweveritsbiologicalroleinT.cruziisstill

unknown.Weshow,byimmunofluorescencelocalization,thatT.cruziDNApolymerasebeta(Tcpol␤)

isrestrictedtotheantipodalsitesofkDNAinreplicativeepimastigoteandamastigotedevelopmental

stages,beingstrictlylocalizedtokDNAantipodalsitesbetweenG1/SandearlyG2phaseinreplicative

epimastigotes.Nevertheless,thispolymerasewasdetectedinsidethemitochondrialmatrixof

trypo-mastigoteforms,whicharenotabletoreplicateinculture.ParasitesoverexpressingTcpol␤showed

reducedlevelsof8oxoGinkDNAandanincreasedsurvivalaftertreatmentwithhydrogenperoxide

whencomparedtocontrolcells.However,thisresistancewaslostaftertreatingTcpol␤overexpressors

withmethoxiamine,apotentBERinhibitor.Curiously,apresumedDNArepairfocuscontainingTcpol␤

wasidentifiedinthevicinityofkDNAofculturedwildtypeepimastigotesaftertreatmentwithhydrogen

peroxide.TakentogetherourdatasuggestparticipationofTcpol␤duringkDNAreplicationandrepairof

oxidativeDNAdamageinducedbygenotoxicstressinthisorganelle.

1. Introduction

Knowledgeregarding mitochondrialDNA repairpathwaysin

protozoanparasitesofmedicalrelevancelikeTrypanosomacruzi,

thecausativeagentof ChagasDisease,isscarceand stillpoorly

understood.T. cruzipossesses onesinglemitochondrion witha

condensedgenomeinacatenatednetworkoftopologically

inter-locked minicircles and maxicircles, known as kinetoplast DNA

(kDNA)[1,2].Whilethousandsofminicircles,comprising90%ofthe

∗Corresponding author at: ICB/UFMG, Department of Biochemistry and Immunology,Av.AntonioCarlos,6627Pampulha,31270-901BeloHorizonte,MG, Brazil.Tel.:+553134092643.

E-mailaddress:crmachad@icb.ufmg.br(C.R.Machado).

network,encodesmallguideRNAsusedinRNAediting[3],dozens

of maxicirclesencoderibosomal RNAsand proteinsinvolved in

mitochondrialenergytransductionascytochromeb,subunitsof

cytochromeoxidaseandsubunitsofNADHdehydrogenase[4].The

maintenanceofkDNAintegrityisvitalforparasitesurvival

dur-ingitslifecycleinsidemammaliancells,wheretheyhavetodeal

withcellulardefenseresponsesthatusuallyinvolveoxygenburst.

Therefore,understandinghowkDNAintegrityismaintainedduring

theparasitelifecycleiscrucialtoidentifypotentialtherapeutical

targetsagainstthisdisease.

Inmammaliancells,reactiveoxygenspecies(ROS)aremostly

producedendogenouslybynormalcellrespiration,andthe

mito-chondrion is believed to be the main intracellular site of ROS

generation[5].Mitochondrialelectrontransportchainactivityis

responsible forthe generationof more than95% of superoxide

Open access under the Elsevier OA license.

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183 (2012) 122–131 123

anionsproducedinsidecells, beingconsideredanabundantand

highly reactive intracellular ROS in mammals [6]. In order to

reducepotentialdownstreamdamage,superoxidesareconverted

tohydrogenperoxide(H2O2)bysuperoxidedismutase.Hydrogen

peroxideis freelydiffusibleacrossmembranes and canoxidize

nucleosidesanddamagebasesinnuclearandmitochondrialDNA

[7].Acommonsite ofoxidativedamageiscarbon8ofguanine,

whichleadstoformationof7,8-dihidro-8-oxoguanine(8oxoG).If

unrepaired,8oxoGcanleadtopotentiallymutagenicandcytotoxic

GC-TAtransversionsduringcellreplication[8].Studiesin

mam-malshaveshownthatthefrequencyof8oxoGonmitochondrial

DNA is 20-fold higher than that observed onnuclear DNA [9],

witha8oxoG/dGrelationbetween0.5to4.24×10−6_[10–12]_._In

addition,themajorityofmutationsintroducedinmitochondrial

DNAcanbedirectlycorrelatedwithquantified8oxoGinmtDNA

[13].Itisreasonabletoconsiderthatmitochondrialoxidative

bur-denmayalsotargetkDNAintrypanosomes.Inordertominimize

the deleterious action of ROS, trypanosomes evolved a unique

biochemicalsystemtodealwithredoxmetabolism.Althoughthey

lackcatalaseandglutathioneperoxidase[14],athioldependent

antioxidantsystemisactivetoeliminatehydrogenperoxideand

associatedbyproducts[15,16].Anti-oxidativeprotectionprovided

bypeculiardetoxifying enzymesistheprimarydefense against

oxidativestressintrypanosomes.Cytosolicandmitochondrial

try-paredoxinperoxidases,involvedprimarilyinmetabolizingH2O2

and short-chain organichydroperoxides by using tryparedoxin

asanelectrondonorviatrypanothione,havebeendescribedinT.

cruzi[17]andT.brucei[18].However,thesesystemsareproneto

saturationandresidualoxidativemoleculescandamagenuclear

andkinetoplastDNA.DNArepairmechanismsprobablyevolvedto

specificallycircumventpre-mutagenicalterationsinthegenome.

SeveralDNA repairputativegeneswerepredictedin T.cruzi

genome[19],includingthoseencodingelementsinvolvedinbase

excision repair (BER) pathway. BER is one of the most

impor-tantpathwaysimplicatedincorrectingoxidativedamageinDNA

[20,21],andcanbereproducedinvitrowithfewpurifiedproteins

fromhumanandEscherichiacoliorigin[22–24].DNArepair

oper-atedbyBERcanoccurviatwosub-pathways.Inshort-patchBER,a

monofunctionalglycosilasecatalyzestheremovalofthedamaged

base,leavinganapurinic/apyrimidinicsite(APsite).Thissubstrate

isrecognizedbyanAP-endonuclease,thatcleavesthe

phosphodi-esterbond5′_to_the_AP_site_[22]_._This_step_is_followed_by_insertion

ofthecorrectnucleotidetothe3′_-OH_end_of_the_incised_AP_site_and

posteriorexcisionoftheremainingsugar-phosphatemoiety(lyase

activity),beingbothprocessescatalyzedbyDNApolymerasebeta.

DNApolymerasebeta(Pol␤)belongstotheXfamilyofDNA

poly-merasesandactsasthemaindRPlyaseeffectorinvolvedinrepair

ofoxidativebaselesions[25–27].

Ontheotherhand,long-patch BERis initiatedwiththe

dis-placementofashortsingle-strandDNA segmentcontainingthe

lesion(usuallyintherangeof2–6nucleotides)carriedoneither

byPol␤orPol␦[28].This“flap”oligonucleotideisthenreleasedby

FEN1endonuclease[29–31].Inbothshort-andlong-patchBER,the

remainingnickissealedbyaDNAligase.Catalyticmeasurements

usingreconstitutedenzymaticsystemsinvitrosuggestedthatthe

rate-limitingstepduringBERistheremovalofdRPcatalyzedbythe

8kDadomainofPol␤[32].Thisway,Pol␤playsanessentialrole

inorientingthelesiontoberepairedthroughshort-orlong-patch

BER,dependingonthetypeofthelesioninvolved.

It is generally accepted that Pol␤ is implicated during the

repairof8oxoGandotheroxidizedpyrimidinesinnuclearDNA

[27,33,34].T.cruzigenomecodesfor twoDNApolymerasebeta

genes(Tcpol␤and Tcpol␤-PAK) that showidentity with

mam-malian Pol␤ [35–38].Previous findingspublished byourgroup

havelocalizedbothpolymerases inside themitochondrionof T.

cruzi[38].Wehaveshownpolymeraseandlyaseactivityforboth

proteinsinvitro[38].However,onlyTcpol␤wasunableto

per-formtranslesionsynthesis(TLS)across8oxoG[38].Forthisreason,

wedecidedtoinvestigateifTcpol␤wasthemainDNApolymerase

operatingduring BER.In thiswork weprovidestrongevidence

that supporta role of Tcpol␤in repairing DNA oxidative

dam-agevia BER and kDNA replication. We reportthat clones over

expressingTcpol␤aremoresensitivetothechain-terminator3′

-azido-3′_{-deoxythymidine}_(AZT),_which_can_be_incorporated_in_the

courseofDNAreplication.Atthesametime,overexpressingcells

aremoreresistanttooxidativestressandcanrepair8oxoGinkDNA

withahigherefficiencywhencomparedtocontrolcells.Inaddition,

epimastigotestreatedwithhydrogenperoxidepresentedaTcpol␤

focuslocalizedatthekinetoflagellarzone.Furthermore,weshow

thatTcpol␤isassociatedwithkDNAantipodalsitesonlyinthose

developmentalstagesfoundinT.cruzilifecyclethatarecapableto

replicate.

2. Materialsandmethods

2.1. Plasmidconstructions

ThevectorusedtogenerateTcpol␤overexpressingparasites

wasconstructedasfollows.First,thecompleteDNApolymerase

betaORFwasretrieved(http://www.genedb.org,accessionnumber

Tc00.1047053503955.20)and PCR-amplifiedfromgenomicDNA

extractedfromT.cruziCLBrenerstrainusingprimerscontaining

XbaI(forward5-TCTAGAATGTTTCGTCGCACA)andEcoRI(reverse

5′_-GAA _TTC _GGG _GTC _GCG _GTT_TTC _CGG)_linkers. _The _product

wasclonedinpGEM-TVector(Promega)followingmanufacturer’s

instructions.Thisconstructwasdigestedandthefragment

liber-atedwasclonedinplasmidpROCK[39]betweenXbaIandXhoIsites.

Twooligonucleotides(5′_AAT_TCG_GCC_AAA_AAG_GCC_and₅′_TCG_AGG

CCTTTTTGGCCG)wereannealedtogethertoformaDNAadaptor

thatallowedligationof3′_end_of_pol_␤_amplicon_with_pROCK_Xho_I

restrictionsite.TocloneatruncatedversionofTcpol␤containing

onlythe8kDadomain,ampliconswereobtainedbyPCR(primers

forward5′_AAG_CTT_ATG_TTT_CGT_CGC_ACG_T_and_reverse₅′_TGA_ATT

CCTTCTCAAGTTCCTC)andclonedintopROCKplasmid

follow-ingthesamestrategydescribedabove.Allplasmidconstructswere

confirmedbyDNAsequencing.

2.2. ProductionofTcpolˇinvitro

Forexpressionand purificationofTcpol␤protein,amplicons

containing5′_-_Eco_RI_and₃′_-_Hind_III_digested_ends_were_cloned_into

pGEM®_-T_Easy_vector_and_subcloned_into_pMAL-g2_(New_England

Biolabs,USA) downstreamfrom themalE geneof E.coli,which

encodesmaltose-bindingprotein(MBP).Subsequentprotein

pro-ductioninE.coliandpurificationwasperformedasdescribed[38].

2.3. InvitroAZTincorporationbyrecombinantTcpolˇ

InhibitionofDNAsynthesisbyrecombinantTcpol␤by3′

-azido-2′_,3′_{-dideoxythymidine}_triphosphate_(AZT)_was_evaluated_by_using

a plasmid treated with DNase as substrate in a reaction that

contained 50mM Tris–HCl, pH7.5, 100mM NaCl, 5␮M dNTPs

and[3_H_}_-dTTP₍₁₀₀_␮_Ci)_and_0.2–4.0_␮_g_of_recombinant_purified

Tcpol␤.After2hat37◦_C,_the_DNA_was_spotted_onto_a_membrane

thatwasthenwashedwith0.5%trichloroaceticacid.The

incorpo-ratedradioactivitywasmeasureinaBeckmanLS6500scintillation

counter.

2.4. Parasitegrowthandtransfection

EpimastigotesfromT.cruziCLBrenerstrainwereculturedat

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124 183 (2012) 122–131

of fetal bovineserum (Cultilab,Brazil), 100U/mL penicillinand

100␮g/mLstreptomycin(Invitrogen,USA).Trypomastigoteswere

obtainedfromsupernatantofinfectedLLCMK2cells(ATCC-CCL7)

and maintainedin Dulbecco modified Eagle’s medium

supple-mentedwith10%fetalbovineserumat37◦_C_and_5%_CO₂_.

Amastig-oteswereobtainedbyincubatingtrypomastigotesinLITmediumat

28◦_C_for₄₈_h._Parasites_were_collected_and_centrifuged_at₂₀₀₀_×_g

for10minandtheresultingpelletwaswashedwith

phosphate-bufferedsaline(PBS) and immediatelyused.Hydroxyurea (HU)

synchronizationwasperformedasdescribedpreviously[41].

Forstabletransfections,atotalof1×108_cells_in_exponential

growthwerewashedandresuspendedin5mLofbuffercomposed

of75%EPB(137mMNaCl,5mMKCl,0.7mMNa2HPO4,6mM

gli-coseand21mMHEPESpH7.5)and25%cytomix[42]containing

50␮gofplasmid thathad beenlinearized withNotI (Promega)

fortargetingto␤-tubulinlocipresentintheparasitegenome.A

plasmidwithouttheresistancecassettewasusedasnegative

con-trolforselection.Afterincubationatroomtemperaturefor15min,

transfections were carried out in 2-mm cuvettes usinga Gene

PulserSystem(Bio-Rad)submittedtotwopulses(10sapart)with

peakdischargeof0.3kVand500␮F. Afterelectroporation, cells

weretransferredinto5mLofLITmediumsupplementedwith10%

fetalbovineserum.Selectionwasappliedafter24hbyaddition

ofG418(200␮g/mL)(Gibco).Selectionoftransfectantswas

con-firmedwhennolivingparasiteswereseeninnegativetransfection

control.Parasiteswereseededinagar-bloodplatescontainingG418

andcloneswereisolated30daysafterincubationat28◦_C.

2.5. NorthernandWesternblots

ToperformNorthernblots,totalRNA(10␮g)wasfractionated

in1.2%agarosegelscontaining5%formaldehyde,blottedontoa

Hybond-N+membrane(GEHealthcare)bycapillarytransfer,and

cross-linkedbyUVirradiation.ADNAprobeof184pbwas

ampli-fiedfromTcpol␤genebyPCRusingprimersforward5′_GGC_TCA

CCTCCGATTATGAAandreverse5′_GTT_GCC_TCG_AGG_TAG_TTT_CG.

Thisprobewasgelpurified,andlabeledwith[a-32P]-dCTPusing

theMegaprimeTMDNAlabelingprotocolfromGEHealthcare.The

membranewashybridizedina50%formamidebufferfor18hat

42◦_C _and _washed _with_2%_SSC/0.1%_SDS _at₆₀◦_C, _as_previously

described[43].T.cruzichromosomalbandswereseparatedbypulse

fieldgelelectrophoresis(PFGE)accordingtoprotocolpreviously

described[44]followedbytransfertoaHybond-N+membrane.

For Western blots, total cellular lysates from 1×109

exponential-phaseepimastigotecellswerepreparedbywashing

cellpelletinphosphate salinebuffer(PBS),and thendissolving

washedpelletsinonevolumeof1×SDSgel-loadingbuffer(50mM

Tris–HClpH6.8,100mMDTT,2%SDS,0.1%bromophenolblueand

10%glycerol)andonevolumeofwater.Cellsweremechanically

lysedbypassagethroughasyringeneedleandboiledfor10min.

Proteins were separated in 8% polyacrylamide gels containing

0.1%SDS usingstandard methods forSDS–PAGE, transferredto

nitrocellulose membranes and incubated with primary (rabbit

polyclonalanti-Tcpolb)diluted1:2000andsecondaryperoxidase

conjugatedantibodies(anti-rabbitIgGdiluted1:2000,Amersham

Biosciences) as described elsewhere. Proteins were detected

using ECL-Plus reagents (Amersham Biosciences) according to

manufacturerinstructionsanddevelopedwithNBT-BCIP.

2.6. Survivalcurves

EpimastigotetransfectedcellsoverexpressingTcpol␤and

con-trolweremaintainedinlogarithmicphase(5×106_cells/mL)_during

5days.Afterthis period,cellsweretreatedwiththreedifferent

doses of 3′_-azido-3′_{-deoxythymidine} _{monophosphate} _(AZT-MP)

(50, 100 and 150␮M) and hydrogen peroxide (200, 300 and

400␮M).Adualtreatmentwasrealizedwith200␮MofH2O2and

10mMofmethoxyaminehydrochloride(MX,Sigma–Aldrich).Cells

wereappropriatelydilutedinPBS1×+erythrosine0.4%and

sur-viving cells werecountedin a Neubauer chamberafter 48hof

incubationat37◦_C.

2.7. Assessmentof8oxoGaccumulationinkinetoplastand nuclearDNA

Tomeasure8oxoGaccumulationinT.cruziDNAcells,weused

aprotocoladaptedfromStruthersetal.[45].Epimastigoteswere

incubatedinthepresenceof200␮Mhydrogenperoxidefor20min

at28◦_C,_washed_twice_with_PBS_and_fixed_with_4%

paraformalde-hyde.Aliquots(20␮L)ofthecellsuspensionweredistributedinto

wellsof8-wellchambered-slides.After1hofincubationat4◦_C,

cells were permeabilized with0.2% Triton X-100,treated with

100␮g/mLRNase A and incubatedwithFITC-conjugated avidin

(5␮g/mL final concentration)for 1hat roomtemperature

pro-tectedfromlight.AfterwashingwithPBS1×andmountedwitha

solutionof9:1glycerol:Tris–HCl,pH9.0,theslideswerevisualized

under a fluorescencemicroscopein a 100x oilimmersion.

Pre-incubationofFITC-conjugatedavidinwith0.5mMof8oxoGresults

in80%decreaseinthefluorescencesignal,whereaspre-incubation

withdGTPhasnosignificanteffectonparasitelabeling.

Fluores-cenceintensitiesfrommanuallydelimitedkinetoplastandnuclear

DNAwereaveragedwiththeImageJprogramandplottedas

fluo-rescencearbitraryunits(averagefluorescenceintensitymeasured

in100cells aftersubtractingtheaveragebackgroundintensity).

Backgroundsignalsweremeasuredin100fields,randomlychosen

ontheslides.

2.8. Immunofluorescenceassays

Indirectimmunofluorescenceexperimentswereperformedby

attaching2×106 _pre-washed_parasites _to_glass_slides,_following

fixation with2% p-formaldehyde in PBS for 20min.Fixed cells

werewashedthreetimeswithPBS,treatedwith0.1%Triton

X-100inPBSfor5min,blocked30minwithPBScontaining1%BSA

andincubatedwiththedifferentaffinitypurifiedanti-Tcpol␤[38]

diluted1:2000inPBScontaining1%BSA.Anti-dihydrolipoamide

dehydrogenase (anti-LipDH),which ispresent inthe

mitochon-drionmatrix,wasraisedinrabbit[46]andwasagiftofDr.Kevin

Tylerandusedat1:1000dilution.Themonoclonalantibody2F6

(mAb2F6)wasusedat1:2dilutionandisspecificforT.cruzi

fla-gellarcalcium-bindingprotein[47].After1h,slideswerewashed

threetimeswithPBSandboundantibodiesrevealedwithanti-IgG

fluorescentconjugatesinthepresenceof0.01mM4′

,6-diamidino-2-phenylindole(DAPI)tostainthenuclearandkinetoplastDNA.

SlidesweremountedwithVectashield(VectorLaboratories)and

observedinNikonE600microscopeormountedinN-propyl

gal-lateandvisualizedbyconfocallaserscanningmicroscope(Zeiss

LSM510META).Alternatively, imageswerecollectedbyusinga

BX61Olympusmicroscope,100×1.4NAobjectivewiththeCell ˆM

software,andprocessedblinddeconvolutionusingtheAutoquant

2.1(MediaCybernetics).Pre-immuneserumorsamplesincubated

withouttheprimaryantibodywasusedasacontrol.

2.9. Transmissionelectronmicroscopy

Protozoawere fixedin 2.5% glutaraldehyde diluted in 0.1M

cacodylatebuffer,pH7.2,for2hatroomtemperatureand

post-fixedin0.1Mcacodylatebuffercontaining1%OsO4,5mMcalcium

chlorideand0.8%potassiumferricyanidefor1h.Then,cellswere

dehydratedinagradedseriesofacetone(30%,50%,70%,90%and

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183 (2012) 122–131 125

Fig.1.OverexpressionofTcpol␤.CellsweretransfectedwithpROCKpol␤andstabletransfectantsselectedinpresenceofG418after30days.(A)Northernblotanalysisusing totalRNAextractedfromepimastigotesinlogarithmicphasederivedfromtwoclones(indicatedby1and2)andnon-transfectedcellsfromCLBrenerstrain(WT).Thelower partshowsthecorrespondingRNAgelstainedwithethidiumbromideasloadingcontrol.(B)Growthcurvesforcontrolcells(WT)andcellsoverexpressingTcpol␤clone1 (C1).Parasitegrowthwasfollowedduring11daysinLITmediumandcellswerecountedinacytometricchamber.Dataarerepresentativeoftwoindependentexperiments. (C)Kinetoplastultrastructuralanalysisofculturedepimastigotecells(day2fromacultureinitiallysetat5×106_cells/mL_LIT_medium)_over_expressing_Tcpol_␤_(left)_and controlcells(right).ArrowspointtopositionofkDNAantipodalsites.(K)Kinetoplast;(Bb)Basalbodies;(Mm)mitochondrialmembranewithcristae.Bars=0.5␮m.

stainedwithuranylacetateandleadcitrateandobservedinaZeiss

900transmissionelectronmicroscope.

3. Results

3.1. Tcpolˇoverexpression

Duetotheimpossibilitytoobtainfunctionalcellswithgenes

knocked-downbyRNAiinT.cruzi,wedecidedtoestablish

epi-mastigotesoverexpressingTcpol␤.Wetransfectedepimastigotes

fromCL Brener strain witha plasmid derived from pROCKNeo

thatcarriedtheentirepol␤gene undercontrolofTcP2␤5′_UTR

andgGAPDHII3′_UTR_flanking_sequences_and_a_constitutive_rRNA

promoter[39].Afterselectionofstabletransfectantclones

resis-tant to G418, we randomly chose two clones for subsequent

experimentation. Northern blot analysis showed an increased

mRNAlevelin theseover expressingcells compared tocontrol

parasites transfected withan empty plasmid (Fig. 1A). To

fur-therevaluateT. cruziDNA polymerasebetaprotein levelsafter

transfection,proteinexpressionwasassessedbyWesternblotin

whole-celllysatesfromcellstransfectedwithemptyvectorand

supposedlyTcpol␤overexpressingparasites(clone1).Ourresults

showthatTcpol␤levelswereelevatedinclone1cellscompared

tothose cells transfected with empty plasmid (Supplementary

Fig.S1).

Similargrowthcurveswereseenbetweencontrolandclone1

cells(Fig.1B). In addition,wecompared kinetoplast

ultrastruc-tural organization of non-treated Tcpol␤ over expressing and

controlcellsbytransmissionelectron microscopy.Our

observa-tionsshowedthatthekinetoplastofover expressingcellswere

similartothoseobservedforcontrolprotozoa,withnodetectable

alterations in kDNA orientation or condensation. Alterations of

mitochondrialmembranesorrepositioningofantipodalsiteswere

notobserved(Fig.1C),indicatingthatTcpol␤overexpressiondid

notmodifymitochondrialDNAorganizationduringthecellcycle

oftransfectedcells.

3.2. TcpolˇoverexpressiondecreasesurvivalinpresenceofAZT

Several purine (e.g. allopurinol) and pyrimidine (e.g. 2′_,3′

-dideoxyinosine and 2′_,3′_{-dideoxyadenosine)} _analogs _can _inhibit

T.cruzi replication[48].Thisis particularlytrueforAZT, which

stronglyinhibitedT.cruziamastigote proliferationcells [48].By

performingcellularsurvival experiments withcontroland over

expressingcellstreatedwithincreasingconcentrationsofAZT,we

foundthatexcessTcpol␤sensitizedthecellsathighdosesofAZT

(Fig.2A),suggesting thatTcpol␤couldbethemainpolymerase

involvedinincorporationofAZTinvivo,asitwasreportedforthe

humanpol␤[49,50].TodemonstratetheimportanceofTcpol␤in

thisprocess,wedesignedinvitroexperimentwithpurified

pro-tein. We showed that AZT can indeed inhibit incorporation of

radioactivethymineinanickedplasmidbyrecombinantTcpol␤

inadose-dependentmanner(Fig.2B).Interestingly,theAZTdose

thatblockedtheactivityofTcpol␤invitro(100␮M)issimilarto

thedosethatislethaltoT.cruziinvivo.Collectively,theseresults

indicatethatTcpol␤canefficientlyincorporateAZTduringkDNA

replication.

3.3. SubcellularlocalizationofTcpolˇisrestricttoantipodalsites inreplicatingcells

TworeplicatingcellularformscanbefoundinT.cruzilifecycle.

Epimastigotesreplicateinsidethegutofthereduviidaebug,while

amastigotesmultiplyintracellularlyinmammalianhost[51].

Try-pomastigotesareinfective,non-replicativeformsthatarereleased

inthebloodstreamofinfectedhostafteramastigotedifferentiation

andcellrupture[51].WeperformedimmunolocalizationofTcpol␤

inculture-derivedCLBrenerwildtypecellsepimastigotes,

intracel-lularamastigotesandtrypomastigotesusingpolyclonalantibodies

againstthepurifiedrecombinantprotein.WefoundthatTcpol␤

ismainlylocalizedinantipodalsitesofreplicativeepimastigotes

(Fig.3I–L)andinextracellularamastigotes(Fig.3E–H)whilein

non-replicativecultured trypomastigotes Tcpol␤ is dispersed inside

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126 183 (2012) 122–131

Fig.2. DNAelongationbyTcpol␤isinhibitedbyAZT.(A)T.cruzisurvivalaftertreatmentwithAZT.CellstransfectedwithemptyplasmidandTcpol␤overexpressing epimastigotes(clone1andclone2)weretreatedwith50,100and150␮g/mLofAZT.Cellswerecountedinacytometricchamberafter72h.Datarepresentthemean±SDof threeindependentexperiments.SignificancewasassessedusingANOVAwithBonferroni’sposttest.*P<0.01.(B)PurifiedrecombinantTcpol␤wasincubatedwithadigested plasmidinthepresenceof5␮MdNTPscontaining3_H-dTTP_and_different_doses_of_AZT._{Radioactivity}_levels_were_measured_and_compared_with_control_reactions._Black_bars indicatecontrolreactionswithTcpol␤orMBPintheabsenceofAZT.WhitebarsarereactionsperformedbyTcpol␤withtheindicateddosesofAZT.

cytoplasm(Fig.3A–D).Tcpol␤mitochondriallocalizationin

try-pomastigotes was furtherevidenced as it colocalized withthe

lipoamidedehydrogenase(LipDH)protein(SupplementaryFig.S2),

amitochondrialmatrixmarker[52].Cellularlocalizationpatterns

forTcpol␤inthethreeparasiteformswereconsistentinmorethan

95%ofthecellsineachexperiment.Theseresultssuggest

partici-pationofTcpol␤duringkDNAreplication,duetoitsassociationto

antipodalsitesinreplicativeformsofT.cruzi,asitwaspreviously

observedforT.bruceiandC.fasciculata[35,53].

3.4. SubcellularlocalizationofTcpolˇfollowsparasitecellcycle

SpecificmorphologicalchangesthatoccurduringT.cruzicell

cycleinvolveashortflagellumthatemergesfromaflagellarpocket

inG2phase,followedbykinetoplastredistributionpriortomitosis

andnucleardivision.Growingoftheemergingflagellumcontinues

untiltwo newdaughtercells areformed[41].Information

con-cerninglocalizationofanyDNApolymeraseenzymethroughout

thecellcycleofkinetoplastidamembersisstilllimited.Inorderto

understandhowDNAreplicationoperatesinT.cruzi,wefollowed

theTcpol␤localizationbyimmunofluorescentapproachduringcell

cycleprogression.WetrackedTcpol␤inthemitochondrionof

epi-mastigoteCLBrenerwildtypecellsbyconfocalmicroscopyusinga

polyclonalantibodyraisedagainstrecombinantTcpol␤.Wewere

abletodetectcellswithmainmorphologicalpatternsthatallowed

ustofollowtheepimastigotecellcycle(Fig.4).CellsinG1/Sphaseof

nuclearcellcyclearecharacterizedbyhavingoneflagellum,

visual-izedwithamonoclonalantibody(mAb25)thatrecognizesa24kDa

flagellarcalcium-bindingprotein,onekinetoplastandonenucleus,

labeledbyDNAstainingby4′_{,6-diamidino-2-phenylindole}_(DAPI).

OncecellsbegintoproceedthoughG1/S,Tcpol␤redistributestothe

antipodalsites(Fig.4A).InearlyG2phase(Fig.4B)whenthenew

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183 (2012) 122–131 127

Fig.4.CellularmorphologicalalterationsandTcpol␤redistributionduringT.cruziepimastigotecellcycle.Thestagesofepimastigotenuclearcellcyclewereidentified basedonthelengthandnumberofflagellae(F),numberofkinetoplasts(K)andnucleus(N)indicatedaboveeachrepresentativeimage.Thenewflagellumemergesfrom cellularpocketwhencellsareinG2stage.Duringmitosistheflagellarpocketsegregatesleadingtocytokinesis,whichgeneratestwoidenticaldaughtercells.Whitearrows indicatethepositionofantipodalsites.WhitearrowheadsindicatethepositionofkinetoflagellarzonewhereTcpol␤seemstoaccumulateinaring-shapedstructureduring cytokinesisandG1phases.Positive(+)andnegative(−)symbolsindicatepresenceorabsenceofTcpol␤inantipodalsites,respectively.SeeSection3.4fordetails.Bar=5␮m.

flagellumhasjuststartedtogrow,muchofTcpol␤wasstillfound

locatedinantipodalsites.Curiously,cellsinlateG2phase(Fig.4C)

inadditiontohavealongergrowingflagellumcomparedtoearly

G2,showedadispersedstainingforTcpol␤,thatwasnomorefound

associatedwithbothantipodalsites.Thispatternpersisteduntil

theendofmitosis,whenkinetoplastandnuclearDNAare

com-pletelyduplicated(Fig.4D).Cellswithtwokinetoplastsandone

nucleuswerenotfoundconsideringthatbothstructuressegregate

almostsimultaneously [41]. Strikingly, duringand immediately

aftercytokinesis,Tcpol␤migratestoaspecificpointbetweenthe

kinetoplastDNAandbasalbodyformingadoughnut-likestructure

(Fig.4E–G).Webelievethatthissitecorrespondstothe

kinetoflag-ellarzone.Aftercytokinesis,Tcpol␤seemstoredistributeagainto

antipodalsitesforasubsequentlyroundofcellreplication.

3.5. Tcpolˇoverexpressionimprovesepimastigotesurvivalin presenceofH2O2

Arole of Pol␤ inDNA repair was extensively demonstrated

in mammals extracts [54], mainly in oxidative damage repair

[21,27,55].ToinvestigatewhetherTcpol␤couldalsoparticipate

inrepairofoxidativedamageinT.cruzi,wemeasuredthesurvival

ofT.cruziepimastigotesoverexpressingTcpol␤thatweregrown

inliquidLITmediumsupplementedwithincreasingconcentration

ofhydrogenperoxide(H2O2),knowntogeneratemultipleoxidized

basesinDNA,including8oxoG,astrongmutagenifnotcorrected

byBER[56].WefoundthatoverexpressingTcpol␤cloneswere

moreresistanttoallconcentrations ofH2O2 tested when

com-paredtocontrol(Fig.5A),supportingthatTcpol␤contributesto

theprocessof DNArepair of oxidativelesionsin epimastigotes

andsuggestingthatexcessTcpol␤couldenhanceBERefficiency.

TobetterclarifytheroleofTcpol␤duringkDNArepair,we

gener-atedparasitesoverexpressingitslyasedomain(8kDa)withoutthe

DNApolymerasedomain.Wetreatedthisculture,aswellasempty

plasmid-transfectedcontrolparasitesandTcpol␤overexpressing

clone1,withhydrogenperoxide(100,200and300␮M).We

veri-fiedthat8kDaoverexpressingparasitesshowedanintermediate

survivalcurvebetweencontrolandTcpol␤overexpressing

para-sites(Fig.5B).Thisdatashowsthatthelyaseactivityisimportant

butnot enoughforcomplete oxidativeDNA lesionrepair. Fully

functionalTcpol␤activityseemstobecrucialinpromotingahigh

efficiencyBERinT.cruziparasitessubmittedtooxidativestress.

We next tested survival of Tcpol␤ over expressing cells in

the presence of H2O2 and methoxyamine hydrochloride (MX).

MX can react with the C’1 of the deoxyribose moieties of

apurinic/apyrimidinicsitespreventingcleavageofthe

phosphodi-esterbondadjacenttotheAP sitebyAPendoribonuclease and,

consequently, inhibiting BER [57,58]. Control and Tcpol␤ over

expressingcellsweresimultaneouslytreatedwith200␮MofH2O2

and10mMofMX,andcellsurvivalwasthenevaluatedafter48h.

WefoundthatinthepresenceofMX,excessofTcpol␤wasnot

ben-eficialforsurvival(Fig.5C).ThisresultsuggeststhatTcpol␤over

expressioncanstimulateBERregardingrepairofoxidativedamage

inkDNAafterH2O2treatment,althoughtheprecisemechanismis

unclear.

Finally, cells were treated with methyl methanesulfonate

(MMS). MMS is a methylating agent that transfers alkylating

adductstoDNA, mainly3-methyladenineand 7-methylguanine

[59,60].Weobservednosignificantdifferencesinsurvivalbetween

Tcpol␤ over expressing and control cells (Fig. 5D). This result

suggeststhatTcpol␤overexpressionis related toresistanceto

oxidativestressandisnotinvolvedinrepairofmethylatingdamage

inT.cruzi.

3.6. 8oxoGisbetterrepairedinkDNAofTcpolˇoverexpressing cell

ConsideringthepossibleroleofTcpol␤inprocessing

mitochon-drialoxidizedbases,wemonitored8oxoGlevelsinthekinetoplast

and nuclear DNA of over expressingPol␤ cells after treatment

withH2O2. Over expressingand control cells wereexposed to

H2O2 andwere thentreated witha FITCconjugateof avidin, a

knownbiomarkerofoxidativeDNAdamage[45].Twohoursafter

treatmentwithH2O2,thefluorescentsignalofFITCinkDNAwas

lowerforTcpol␤overexpressingcell(clone1)comparedtocontrol

(Fig.6AandC).Thisdifferencepersistedatlast24haftertreatment.

Incontrast,measurement ofFITCsignalinnuclearDNAshowed

similarlevelsofoxidativedamagebothincontrolandTcpol␤over

expressingclone2hand24hafterH2O2treatment(Fig.6BandC).

ThesedatasupportthatexcessTcpol␤favorstherepairof8oxoG

inkDNA.

3.7. Tcpolˇtriplefociformationaftertreatmentofcultured epimastigoteswithH2O2

SeveralmammalianDNArepairproteinslikePol␤andOGG1can

associateindiscretefociaftergenotoxicstress,includingoxidative

stress[61,62].Inthesecases,thefocipersistforadefinedperiod

oftimeandthenaredismantled,whichsuggestsformationofa

repairosomethatremainsactiveonlyduringDNArepairprocesses.

Therefore,wedecidedtoimmunolocalizethenativePol␤fromT.

cruziaftertreating wildtypeepimastigotecells fromCLBrener

strainwithH2O2.Sixhoursafterexposuretooxidativestress,we

wereabletodetectformationofathirdfocusofTcpol␤

addition-allytothattwopreviouslyseenadjacenttokDNAantipodalsitesin

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128 183 (2012) 122–131

Fig.5. Tcpol␤isinvolvedinoxidativestressresponseinT.cruzi.(A)CellstransfectedwithemptypROCKplasmidandTcpol␤overexpressingclones(clone1andclone2) weretreatedwith200,300and400␮MofH2O2.Cellswerecountedinacytometricchamberafter48h.(B)Emptyplasmid-transfectedcells(pROCK),8kDaandTcpol␤ (clone1)overexpressingparasitesweretreatedwith100,200and300␮MofH2O2.Cellswerecountedinacytometricchamberafter48h.(C)Simultaneoustreatmentof Tcpol␤overexpressingandcontrolcellswith200␮MH2O2and10mMmethoxyamine(MX).Cellswerecountedafter48hinacytometricchamber.(D)T.cruzisurvival aftertreatmentwithMMS.Cellstransfectedwithemptyplasmid(pROCK)andTcpol␤overexpressingclonesweretreatedwithMMSrangingfrom0.001to0.003%.Cells werecountedinacytometricchamberafter72h.Experimentaldatarepresentthemean±SDofthreeindependentexperiments.SignificancewasassessedusingANOVA withBonferroni’sposttest.*P<0.01.

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183 (2012) 122–131 129

Fig.7.AccumulationofTcpol␤inepimastigotecellstreatedwithhydrogenperoxide.FixedcellsimmunostainedforendogenousTcpol␤inepimastigoteformsofT.cruzi aftertreatmentwith100mMH2O2.Representativeimageswereselectedfromatotalof100cellsanalyzedineachtimepoint.Localizationpatternsofuntreatedcells(A–D), andofcells1h(E–H),6h(I–L)and24h(M–P)aftertreatmentareshown.MergeshowsbothnuclearandkinetoplastDNAinblueandTcPol␤inred.Thewhitearrowindicates aTcpol␤focusformationobservedafteroxidativetreatment.Bar=5␮m.

clearlypositionedoutsidethekDNAregion,andwebelievethatit

isprobablylocatedinkinetoflagellarzone(KFZ).Thisfocuswasnot

detectedinnon-treatedcells(Fig.7AandB)anditsdetectionwas

notpossibleat1hand24hafterH2O2treatment(Fig.7E–Hand

M–P,respectively).Thisextrafocuswasnotobservedaftertreating

cellswithmonofunctionalalkylatingagentmethoxyamine(MMS)

(datanotshown).

4. Discussion

Maintenance of kDNA integrity relies on a complex process

involving a large collection of enzymatic factors and an

intri-catedynamicsof minicircleand maxicirclenetworkreplication.

Althoughourknowledgeabouttheseprocesseshasbeenimproved

considerable,littleisknownabouthowoxidizedbasesinkDNAof

trypanosomesarerecognizedandrepaired.UsingT.cruziasmodel,

wefocusedontheroleofTcpol␤,akeycomponentofBERrepair

pathwayinpreventingthedeleteriouseffectsof8oxoGinthe

mito-chondrialgenomeofthisparasite.

ParasitesoverexpressingTcpol␤werefoundmoresensibleto

AZTregimencomparedtocontrolcells.Thisdiminishedsurvival

couldbeattributedtoanincreasedincorporationofAZTbyexcess

ofTcpol␤intokDNAduringmitochondrionreplication,asitwas

previouslyshowninmammaliancells.

By immunolocalization assays we showed that Tcpol␤ is

directedtothekinetoplast antipodalsites inepimastigotes and

amastigotes,whileintrypomastigotesitisspreadthroughoutthe

cellbody,mostlikelyinsideitssinglebranchedmitochondrion.The

biochemicalprocessessupposedtooccuratantipodalsites,mainly

fromstudiesinT.brucei,includedegradationofRNAprimersstill

annealedtothenewlyreplicatedminicirclesbySSE1(after

migra-tionofminicirclesfromthekinetoflagelarzone)andgaplinking

byDNApolymerasebeta[35].Althoughourworkdonotformally

demonstratetheroleofDNApolymerasebetainT.cruzi,theclear

associationbetweenreplicativestatusandpatternofsubcellular

localizationissuggestiveofaroleofthispolymeraseinkDNA

dupli-cation,whenTcpol␤couldfillthegapsbetweenOkasakifragments

afterreplicatingtheminicircles. Similarpatternsof localization

intheantipodalsitesofkDNAwereobservedinT.bruceiandC.

fasciculata[35,53].ThisresultisdifferentforL.infantum,which

possessanuclearPol␤[37].Intriguingly,noTcpol␤signalcould

bedetectedinthenucleusofT.cruzi.Webelievethatother

poly-merases,likePol␧andPol␦,couldpromotefunctionalredundancy

inshortand long-patchnuclearBER,as demonstratedin

mam-maliancellextractsdeficientinPol␤[34,63].

InordertoelucidatetheroleofTcpol␤inkDNArepair,

hydro-genperoxidewasusedasgenotoxicagentinsurvivalcurvesbyits

capabilitytoinduceoxidativeDNAdamage[64,65].Using

differ-entdosesofH2O2weverifiedthatepimastigotesoverexpressing

Tcpol␤showedincreasedsurvivalwhencomparedtocontrolcells.

However,parasites over expressingatruncated formofTcpol␤

(8kDadomainonly)presentedanintermediateresistant

pheno-typebetweencellstransfectedwithemptyplasmidandclone1

parasites.Theresultssuggestthatthe8kDadomainalonecould

participateinBERremovingthedRPandotherpolymerases,such

pol␧,couldsubstituteTcpol␤toinsertnucleotides,butwithlower

efficiency.Inthissense,fullyfunctionalTcpol␤enzymeis

manda-torytoallowBERtoefficientlyremoveoxidativelesionsinT.cruzi

kDNA.

WebelievethatBER couldbethemain DNArepairpathway

actingondetoxificationofperoxidesinthiscondition,considering

thatinitialsanitizationofH2O2bytryparedoxinperoxidaseis

effi-cientinlowdosesrangingfrom2.5to20␮MofH2O2andweused

higherdoses(200–400␮MH2O2)insurvivalcurves[66].Theability

ofMX,aspecificshort-patchBERblockingagent,tosensitizeover

expressingcellstoH2O2suggestsspecificparticipationofTcpol␤

inimprovementofsurvivalwhenitisoverexpressedinsidecell.In

addition,survivalexperimentsperformedwithMMSdidnotshow

anysignificantdifferencebetweenTcpol␤overexpressingand

con-trolcells.MammalianBERisinvolvedinrepairofalkylatingdamage

causedbyMMS,includingthecytotoxicadducts3-methyladenine

and7-methylguanine[59,60,67,68].PurinesdamagedbyMMSare

recognizedby3-methyladenineDNAglycosylase(AAG)inhumans

[69].TheobservedinsensitivityofT.cruzitoMMScanbedueto

inexistenceofafunctionalhomologueofAAGinT.cruzi

mitochon-driatoinitiateBER.MMSalsoinducehomologousrecombination

bygeneratingsingleand double-strand breaks[70,71]. Another

possibleinterpretation is that MMS is incapable tosensitize T.

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130 183 (2012) 122–131

processinthisorganism[44].ItisalsopossiblethatMMSdamage

is mainly repaired by long-patch BER in T. cruzi, considering

thatMMSwasadministratedtologarithmicgrowthphase cells,

where short-patch BER predominates. Additional experiments

are beingperformedto elucidate resistanceof T. cruzitoMMS

treatment.

WebelievethatTcpol␤overexpressingcellsresistancetoH2O2

isprobablyduetoenhancedDNArepairofoxidizednucleotidesin

kinetoplast,aswesawdepletedlevelsofoxoguanineinthis

struc-turecomparedtocontrolcells.Depletedoxoguaninelevelsinover

expressingclone1after2hcanindicatehigheractivityofBERin

thesecells,probablystimulatedbyanexcessofTcpol␤.After24h,

8oxoGlevelsincreasedinbothcontrolandoverexpressingcells,

althoughlesspronunciatedinoverexpressingcells.Thisisprobably

duetoincorporationofoxodGderivingfromoxidizednucleotide

poolduringcellreplication.Wewerenotabletodetectdifferences

inaccumulationof8oxoGinnuclearDNAfrombothcells,which

couldindicatestrictlystimulationofmitochondrialBERbyTcpol␤

overexpression.TheseresultssuggestthatotherDNArepair

path-waysinT.cruzithatcanrecognizeoxidativedamageareactiveinthe

nucleus.TakentogethertheseevidencessupportaroleofTcpol␤

inrepairof8oxoGinkDNAofT.cruzi.

Astrikingfindingisthatgenotoxictreatmentofepimastigotes

withhydrogenperoxideinducesmobilizationofTcpol␤toadefined

focus,inadditiontothetwofocipreviouslyseenintheantipodal

sitesintheabsenceoftreatment.ItwasshowninHeLacellsthat

BERproteinslikePol␤,OGG1andXRCC1canbedirectedtorepair

fociafterinducinglocaloxidativestress[61,62].Wespeculatethat

thisnewfocusislocatedinthekinetoflagelarzonewhereminicircle

replicationtakesplace,consideringtheunavailabilityof

mitochon-drial markersthat couldbeused torefinethis localization. We

hypothesizethatwiththehigherlevelofoxidativestressimposed

byH2O2treatment,Tcpol␤couldbedirectedwithinthisareato

participateofextratemporalBEReventsinaspecializedfocus

out-sidethekinetoplastduringpossibleasynchronicDNArepairevents

thatnotoccurduringG1/S.

ThisisthefirsttimethatamemberofXfamilyofDNA

poly-merases is immunolocalizedin trypomastigotes of T. cruzi.The

diffuselocalization of Tcpol␤seen intrypomastigote cells gave

uspreciousinsightsabouthowaBERpolymerasecouldpromote

protectionagainstgenotoxicstressinthisstage.Thisisconsistent

withthefactthattrypomastigotesdo notreplicateDNA,

releas-ingTcpol␤tobedirectedtoantipodalsitesinthisstage.Probably

thestoichiometryofTcpol␤insidemitochondrionistightly

reg-ulated.ArapidsupplyofTcpol␤toantipodalproteincomplexes

duringdifferentiationfromtrypomastigotestoepimastigotesand

amastigotes couldbe efficiently achieved byinternal

mobiliza-tioninsteadofbeingdegradedandproducedwhenitisneeded.

ThehypothesisofTcpol␤degradationcanbealsoconsideredand

additionalexperimentsarerequiredtobetterunderstandofthis

process.

PreviousstudiesinvolvinglocalizationofDNArepairproteins

intrypanosomatidspresentedstaticmodelsofsubcellular

kineto-plastorganizationofthereplicationmachinery[1].Inthesemodels,

pol␤isclearlylocalizedinantipodalsites[35,38,72].Inthis

arti-clewealsoshowedthatduringepimastigotecellcycleTcpol␤is

mobilizedinsidemitochondrion,anditseemsthatthepolymerase

isonlyrequiredinantipodal sitesin G1toSphasewhen

mito-chondrial DNAis beingsimultaneouslyreplicated and repaired.

Thisis thefirsttimethatredistribution ofaDNArepairprotein

alongcellcycleisshownforakinetoplastidamember.Itis

pos-siblethat this relocation todifferent subcellulardomains must

beanadditionalenzymatic controlfor Tcpol␤thattheparasite

usestoimproveitsBERrepairefficiencyduringgenome

replica-tion.Additionalapproachesmustbetakentobetterelucidatethis

phenomenon.

In summary, our experimental data points toward a role

of Tcpol␤ from T. cruzi in conferring resistance to genotoxic

oxidative stress. Acquisitionof a deeper understanding

regard-ing involvement of this DNA polymerase and BER pathway in

repairofoxidativelesionswillcertainlyallowbetterexperimental

approachesandinterventionduringtreatmentofChagasDisease.

Acknowledgments

This study was supported by CNPq-Brazil

(MCT/CNPq/MS-SCTIE-DECIT25/2006-EstudodeDoenc¸asNegligenciadas),PRONEX

andFAPEMIG.TheresearchfromS.M.TeixeiraandC.R.Machado

wassupported,inpart,byanInternationalResearchScholarsGrant

fromtheHowardHughesMedicalInstitute.WearegratefultoKátia

BarrosoGonc¸alvesandNeuzaAntunesRodriguesfortechnical

sup-port.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,atdoi:10.1016/j.molbiopara.2012.02.007.

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