Phosphoglycolate-terminated DNA Double Strand Breaks by Artemis Nuclease* S

13 Pages·2007·0.68 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.282,NO.6,pp.3547–3558,February9,2007
PrintedintheU.S.A.
(cid:1)
Processing of 3 -Phosphoglycolate-terminated DNA Double
Strand Breaks by Artemis Nuclease*□S
Receivedforpublication,August14,2006,andinrevisedform,October31,2006 Published,JBCPapersinPress,November22,2006,DOI10.1074/jbc.M607745200
LawrenceF.Povirk‡,TongZhou‡,RuizheZhou‡,MortonJ.Cowan§,andStevenM.Yannone¶1
Fromthe¶LifeSciencesDivision,LawrenceBerkeleyNationalLaboratory,Berkeley,California94720,the‡Departmentof
PharmacologyandToxicology,MasseyCancerCenter,VirginiaCommonwealthUniversity,Richmond,Virginia23298,
andthe§DepartmentofPediatrics,UniversityofCalifornia,SanFrancisco,California94143
The Artemis nuclease is required for V(D)J recombination deficiency(SCID)2inhumans,designatedRS-SCID(radiation-
andforrepairofanasyetundefinedsubsetofradiation-induced sensitiveSCID)(1)orSCIDA(AthabascanSCID)(2).TheArte-
DNAdoublestrandbreaks.ToassessthepossibilitythatArte- misproteinisanucleasethatisactivatedbyDNA-dependent
misactsonoxidativelymodifieddoublestrandbreaktermini,its protein kinase (DNA-PK) and is required for the opening of
activity toward model DNA substrates, bearing either 3(cid:1)-hy- hairpin ends formed during V(D)J recombination (3), thus
droxyl or 3(cid:1)-phosphoglycolate moieties, was examined. A accounting for the SCID phenotype associated with Artemis
3(cid:1)-phosphoglycolatehadlittleeffectonArtemis-mediatedtrim- deficiency.SCIDAandRS-SCIDfibroblastsareradiation-sen-
ming of long 3(cid:1) overhangs (>9 nucleotides), which were effi- sitivebutfailtorepaironlyasmallfractionofradiation-induced
cientlytrimmedto4–5nucleotides.However,3(cid:1)-phosphogly- DNA double strand breaks (DSBs) (4–6). In vitro, activated
colatesonoverhangsof4–5basespromotedArtemis-mediated Artemisremoves5(cid:3)overhangsfromDNAendsandshortens3(cid:3)
removalofasingle3(cid:1)-terminalnucleotide,whileatleast2nucle- overhangs(7),raisingthepossibilitythatduringDSBrepairin
otidesweretrimmedfromidenticalhydroxyl-terminatedsub- vivo, Artemis may trim overhangs that otherwise cannot be
strates. Artemis also efficiently removed a single nucleotide processedtogiveligatableends.
fromaphosphoglycolate-terminated3-base3(cid:1)overhang,while AbouthalfofDNAbreaksinducedbyionizingradiationbear
leaving an analogous hydroxyl-terminated overhang largely 3(cid:3)-phosphoglycolate (3(cid:3)-PG) termini in various contexts (7)
intact. Such removal was completely dependent on DNA-de- that must be removed in order to allow gap filling by DNA
pendentproteinkinaseandATPandwaslargelydependenton polymerases (cid:1) and (cid:2) and ligation by DNA ligase IV (8).
Ku, which markedly stimulated Artemis activity toward all 3(cid:1) Although tyrosyl-DNA phosphodiesterase (TDP1) is the only
overhangs.Together,thesedatasuggestthatefficientArtemis- identified enzyme capable of processing 3(cid:3)-PGs on 3(cid:3) over-
mediated cleavage of 3(cid:1) overhangs requires a minimum of 2 hangs(9),TDP1mutantcellsshowonlymarginalradiosensitiv-
nucleotides,oranucleotideplusaphosphoglycolate,3(cid:1)tothe ity(10),suggestingtheexistenceofanalternativepathwayfor
cleavagesite,aswellas2unpairednucleotides5(cid:1)tothecleavage processingoftheselesionsinvivo.
site. Shorter 3(cid:1)-phosphoglycolate-terminated overhangs and BecauseArtemis-deficientcellsexhibitsignificantsensitivity
blunt ends were also processed by Artemis but much more toionizingradiation,yethaveonlyasmallDSBrepairdefect,we
slowly.ConsistentwitharoleforArtemisinrepairofterminally assessed the possibility that Artemis functions in processing
blockeddoublestrandbreaksinvivo,humancellslackingArte- overhanging3(cid:3)-PGtermini.WepurifiedrecombinantArtemis
misexhibitedhypersensitivitytox-rays,bleomycin,andneocar- andexaminedtheactivityofthisproteinonavarietyofoligo-
zinostatin, which all induce 3(cid:1)-phosphoglycolate-terminated mericandplasmidsubstratesbearing3(cid:3)-overhangs.Wefurther
doublestrandbreaks. investigated the relevance of Artemis to the repair of 3(cid:3)-PG
terminatedDSBsbymeasuringthetoxicityofdrugsknownto
inducesuchbreaksinnormalandArtemis-deficientcells.
TheArtemisgeneticlocuswasidentifiedbyvirtueofitsasso-
EXPERIMENTALPROCEDURES
ciationwithaformofB(cid:1)T(cid:1)NK(cid:2)severecombinedimmune
Protein Expression and Purification—Artemis expression
constructs were derived from full-length Artemis cDNA as
*ThisworkwassupportedbyNCIGrantCA40615fromNationalInstitutesof described previously (2). Full-length Artemis protein was
Health,DepartmentofHealthandHumanServicesandtheOfficeofSci- purified from SF9 insect cells infected with recombinant
ence,UnitedStatesDepartmentofEnergyContractDE-AC03-76SF00098 baculovirus generated by subcloning Artemis cDNA into
throughtheLowDoseRadiationResearchProgram,andNationalInsti-
pFASTBAC-HT(Invitrogen).Briefly,theamino-polyhistidine-
tutesofHealthGrantsAI28339andHL58842.Thecostsofpublicationof
thisarticleweredefrayedinpartbythepaymentofpagecharges.This taggedArtemiswasextractedandpurifiedusingimmobilized
articlemustthereforebeherebymarked“advertisement”inaccordance metalaffinitychromatographywithstandardprotocols(nickel-
with18U.S.C.Section1734solelytoindicatethisfact.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplementalFigs.1–4. 2The abbreviations used are: SCID, severe combined immune deficiency;
1To whom correspondence should be addressed: Life Sciences Division, DNA-PK,DNA-dependentproteinkinase;DNA-PKcs,DNA-PKcatalyticsub-
Dept.ofMolecularBiology,LawrenceBerkeleyNationalLaboratory,Mail unit;DSB,doublestrandbreak;PG,phosphoglycolate;TDP1,tyrosyl-DNA
Stop74-157,1CyclotronRd.,Berkeley,CA94720.Tel.:[phone];Fax: phosphodiesterase;HPLC,highpressureliquidchromatography;BrdUrd,
510-486-6816;E-mail:[email protected]. bromodeoxyuridine.
FEBRUARY9,2007•VOLUME282•NUMBER6 JOURNALOFBIOLOGICALCHEMISTRY 3547
This is an Open Access article under the CC BY license.
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
nitrilotriacetic acid; Amersham Biosciences). Fractions con- EDTAbyheatingamixtureoflabeledoligomeranda1.5-fold
tainingArtemisweredialyzedinto50mMHEPES,pH7.5,10% excessofunlabeledcomplementto80°Cfollowedbyslowcool-
glycerol,2mMEDTA,1mMdithiothreitol,0.01%NonidetP-40, ing to 10°C over a period of 3 h. Internally labeled plasmid
20 (cid:1)g/ml phenylmethylsulfonyl fluoride, 1 (cid:1)g/ml aprotinin, substrateswithvariousoverhangswereconstructedbyligating
pepstatinA,andleupeptin(HCB),containing0.1MNaCl.The 3(cid:3)-PG, 3(cid:3)-phosphotyrosyl, or 3(cid:3)-hydroxyl oligomers (9–24
affinity-purified protein was loaded onto a Mono Q column basesinlength)intoplasmidswithan11-base5(cid:3)overhang,as
(Amersham Biosciences) and eluted with a linear gradient of described(13).Eachplasmidwasgel-purifiedandeluted,and
100–500 mM NaCl in HCB. Aliquots of Artemis-containing theconcentrationwasdeterminedfromthe260nmpeakofthe
fractionsweresnap-frozenandstoredat(cid:1)70°C.Proteinfrom absorbancespectrum.
thesealiquotswassubjectedtotandemmassspectrometryand Nuclease Assays—Reaction mixtures (10 (cid:1)l) containing 25
identifiedasArtemis.Endonucleaseandexonucleaseactivities mMTris-HCl,pH8,25mMNaCl,10mMMgCl ,1mMdithio-
2
wereverifiedbystandardbiochemicalassays,andaliquotswere threitol, 0.25 mM ATP, 50 (cid:1)g/ml bovine serum albumin, and
discardedafterfourorfewerfreeze/thawcycles.Ku70/80was either5nMoligomericsubstrateor1nMplasmidsubstratewere
purifiedfrominsectcellscoinfectedwithamixtureofrecom- prepared at 22°C. In some cases, a blunt-ended double-
binant baculovirus harboring the human KU70 and KU80 stranded35-mer(4nMto1(cid:1)M)wasalsoincluded(3).Kuwas
genes, and DNA-PKcs was purified from HeLa cells, both as added,andthemixturewasimmediatelyvortexedandbriefly
described previously (11). All protein concentrations were centrifuged.DNA-PKcswasthenaddedfollowedimmediately
determinedbyBradfordassaysusingbovineserumalbuminas byArtemis,andthereactionwasmixedbypipetingandplaced
aproteinstandard(Bio-Rad),andproteinpuritywasevaluated in a 37°C bath. Reactions with oligomeric substrates were
by Coomassie Blue-stained SDS-polyacrylamide gels. When stoppedbyadditionof10(cid:1)lofformamidecontaining20mM
necessary, proteins were diluted immediately before use in EDTA, and the DNA was heat-denatured and analyzed on
reactionbufferlackingATPeitheronice(Artemis,DNA-PKcs) sequencinggels.Reactionscontainingplasmidsubstrateswere
orat22°C(Ku). stoppedbyadditionof20(cid:1)lof10mMEDTA,0.45Msodium
DNA Substrates—Oligonucleotides were purchased from acetate, 100 (cid:1)g/ml tRNA, followed immediately by phenol
QiagenorIntegratedDNATechnologies.Alllabeledoligomers extractionandethanolprecipitation.DNAwascutwithAvaI
werepurifiedbygelelectrophoresisfollowedbyreverse-phase (20units,6h,37°C)orTaq(cid:4)I(hereafterreferredtoasTaqI;20
HPLC.Theyieldofeacholigomerwasdeterminedbyintegrat- units,4h,65°C)in50(cid:1)lofthebuffersuppliedbythevendor
ingtheabsorbanceat260nmrecordedonanin-lineUVmon- (NewEnglandBiolabs).Insomecases0.25mMCoCl and0.1
2
itor.Togenerate3(cid:3)-PGoligomerswiththesequenceCGAGG- mM ddGTP were subsequently added, and the samples were
AACGCG(A )CG (0 (cid:3) n (cid:3) 4), 5(cid:3)-32P-end-labeled oligomers treated with 20 units of terminal deoxynucleotidyltransferase
n
CGAGGAACGCG(A )CGCCC were treated with bleomycin (NewEnglandBiolabs)for1hat37°C.DNAwasprecipitated,
n
plus H O , and the desired 14–17-base 3(cid:3)-PG products were dissolved in formamide, heat-denatured, and analyzed on
2 2
isolatedfromasequencinggelandpurifiedbyHPLC(12).The sequencinggels.TheAvaIandTaqIcleavagesitesdifferbyone
terminalstructureandpurityofthe14-base(n(cid:4)1)3(cid:3)-PGoli- nucleotide; however, as expected these two enzymes always
gomerwasverifiedbyelectrospraymassspectrometryandfrag- gaveessentiallyidenticalresultsinparallelexperiments.
mentation-basedchemicalsequencing.Treatmentoftheother Cell Lines and Proliferation Assays—Fibroblast lines were
(n(cid:4)0and2–4)oligomerswithbleomycinlikewiseresultedin establishedfromskinbiopsiesfromSCIDApatients04and05
efficientsite-specificcleavageatthe-CGCCCsitenearthe3(cid:3) and an unrelated immunologically normal individual (AK).
end,asjudgedbytheappearanceofaprominentbandwiththe Early passage cultures were immortalized by introduction of
expectedmobility(supplementalFig.1).Uponextractionfrom humantelomerasereversetranscriptasecDNAusingmethods
thegel,eachsucholigomerelutedasasinglewelldefinedpeak previouslydescribed(6,11).Cellswereeitherirradiatedusinga
fromreverse-phaseHPLC.Theterminalstructureandpurityof Pantak(cid:3)x-raygeneratoroperatingat320kV/10mAwith0.5
each putative 3(cid:3)-PG oligomer was confirmed by quantitative mm copper filtration or exposed to genotoxic drugs for 1 h,
conversiontothecorresponding3(cid:3)-phosphateand3(cid:3)-hydroxyl washedthreetimeswithPBS,andthenlabeledbytheaddition
oligomers by treatment with tyrosyl-DNA phosphodiesterase of fresh medium containing 10 (cid:1)g/ml bromodeoxyuridine
and polynucleotide kinase/phosphatase, which in every case (BrdUrd)(Sigma)for24–35h.Cellswereharvested,fixed,and
inducedtheexpectedshiftsinelectrophoreticmobility(supple- stainedusingstandardprocedures,andcellcycledistribution
mental Fig. 1). An analogous oligomer (n (cid:4) 1) bearing a and BrdUrd incorporation were analyzed with a Beckman-
3(cid:3)-phosphotyrosylterminuswaspurchasedfromMidlandCer- CoulterEPICSXL-MCLflowcytometerusingXLDataAcqui-
tifiedReagents.The9-and11-base3(cid:3)-PGoligomersCGAGG- sitionsoftwareandWinMDI2.8orFlowJo8.1softwarepack-
AACG and CGAGGAACGCG were similarly prepared as ages. The fraction of proliferating cells (F) was calculated by
describedpreviouslyandtheirstructuresverifiedbymassspec- scoringthepercentofintactcellsstainingpositiveforBrdUrd
trometry (12). The 36-base 3(cid:3)-PG or 3(cid:3)-hydroxyl oligomers andnormalizingtotheuntreatedcontrolofthesamecellline
werepreparedbyligatingthecorrespondinglabeled14-mersto ((cid:5)10,000eventswerescoredforeachpoint).Toquantitatively
the 22-mer GCCATGTACTTGGATGATCTAT in the pres- comparethetoxicityofeachagenttowardSCIDAandnormal
enceofthecomplementary20-merGCGTTCCTCGATAGA- cells,relativetoxicityateachradiationdoseordrugconcentra-
TCATC and were again gel/HPLC-purified. Partial duplexes tion was calculated as ln(F1)/ln(F2), where F1 and F2 are the
were annealed in 10 mM Tris-HCl, pH 8, 0.1 M NaCl, 1 mM proliferating fractions, normalized to untreated controls, for
3548 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER6•FEBRUARY9,2007
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
SCIDAandnormalcells,respectively.Thisparameterwasrel-
ativelyconstantoverarangeofconcentrationseveninthecase
ofbleomycin,whichtypicallyshowsadistinctupwardconcav-
ityintheresponse.
RESULTS
TrimmingofLong3(cid:3)-PG-terminatedOverhangsbyArtemis—
DSBsinducedbyradiationandotherfree radical-based toxins
commonly bear 3(cid:3)-PG termini (7, 14–16), which block poly-
merase and ligase activities as well as most human exonucle-
ases.TheArtemisnuclease,whichreportedlyshortenslong3(cid:3)
overhangs,couldtherebyresolvePGandotherblockedtermini
on3(cid:3)overhangsofDSBs.Todeterminewhethertheendonu-
cleolyticactivityofArtemiswasaffectedbythepresenceofPG
termini,aninternallylabeled3(cid:3)-PGor3(cid:3)-hydroxyl-terminated
36-merwasannealedto21-,23-,and27-basecomplementary
strands to yield substrates with 15-, 13-, and 9-base 3(cid:3)
overhangs.
As expected, Artemis treatment of the 3(cid:3)-hydroxyl sub-
strates resulted in efficient trimming of overhangs (Fig. 1A)
(17). The 15-base (36/21 substrate) and 13-base (36/23 sub-
strate) overhangs were each predominantly shortened to a
5-base overhang, yielding 26- and 28-base products, respec-
tively. The 9-base overhang (36/27p substrate) was trimmed
predominantly to a 4-base overhang (31-mer) rather than
5-base overhang. The PG-terminated 15- and 13-base over- FIGURE1.EffectofsubstratelengthandPGterminioncleavageof3(cid:1)
hangs were likewise trimmed predominantly to 5-base over- overhangsbyArtemisnuclease.Allsamplesweretreatedwith65nMKu,55
nMDNA-PKcs,and/or240nMArtemis,asindicated.A,internallylabeledoligo-
hangs,whereasthe9-baseoverhangwastrimmedtoa4-base
mericsubstrateswitha17-base(36/19),15-base(36/21),13-base(36/23),or
overhang. These cleavage patterns and levels of activity are 9-base(36/27p)3(cid:3)overhangandbearinga3(cid:3)-hydroxylterminusweretreated
essentiallyidenticaltothoseobtainedwiththeanalogous3(cid:3)-hy- withArtemisplusDNA-PK.B,analogousinternallylabeledsubstrateswiththe
indicatedPG-terminated(F)3(cid:3)-overhangsweretreatedwithArtemisplus
droxyloverhangs(compareFig.1,AandB),indicatingthatPG DNA-PK. C, end-labeled substrates with 13-base (36/23), 19-base (42/23),
moieties several bases from the cleavage site had little or no 25-base(48/23),and29-base(48/19)3(cid:3)overhangsweretreatedwithArtemis
and/orDNA-PK.Replicateexperimentsyieldedessentiallyidenticalresults.
effectontheefficiencyorspecificityofArtemis-mediatedDNA The 27p oligomer was 5(cid:3)-phosphorylated, but the other complementary
cleavage.Inallcases,38–60%ofthesubstratewascleaved,with strandswerenot.NotethatthesubstratesinAandBareinternallylabeled14
themajorproductaccountingfor50–66%ofthetotal. basesfromthe3(cid:3)end.Thus,theArtemiscleavageproducts,whichbearboth
5(cid:3)-and3(cid:3)-hydroxyls,runafullnucleotidepositionmoreslowlythanMaxam-
A substrate composed of a 19-mer annealed to the labeled Gilbertmarkersofthesamelength,whichbear5(cid:3)-and3(cid:3)-phosphates;for
36-mer, with 19 bp of duplex DNA and a 17-base overhang, example,the31-basecleavageproductfromthe36/27pduplexcomigrates
withthe32-basemarkergeneratedbychemicalcleavageoftheGatposition
sustained at least 3-fold less Artemis-mediated cleavage than
33inthesequence(...CGCGACG).D,purityofproteinsusedinArtemiscleav-
theothersubstratesandshowedalteredspecificity(Fig.1A).To agereactions.RecombinantArtemis(10(cid:1)g),recombinantKu(10(cid:1)g),and
determinewhetherthisinefficiencywasbecauseofthe19-bp DNA-PKcs(5(cid:1)g)purifiedfromHeLacellsweresubjectedtodenaturingPAGE
andstainedwithCoomassieBlue.Leftmostlanecontainsmolecularsizemark-
duplexbeingtooshorttoaccommodatetheKu-DNA-Artemis
ers(kDa).
complex,orthe17-baseoverhangbeingtoolongtoalloweffi-
cientcleavage,wepreparedsubstrateshavingthesame23-bp DNAforproperbindingandalignmentofthenucleasecom-
duplexDNAanddifferingonlyinthelengthofthe3(cid:3)single- plexisnotsurprising.
stranded overhangs, 13, 19, and 25 bases. Artemis nuclease ControlreactionshereandthroughoutrevealthatArtemis,
cleaved all three substrates, with comparable efficiency and Ku,andDNA-PKcsproteinpreparationsarefreeofsignificant
essentiallyidenticalspecificity,withthedominant5-baseover- contaminatingnucleases.ACoomassieBlue-stainedpolyacryl-
hang(28-mer)productaccountingfor44–52%oftotalcleavage amide gel showing the purity of preparations of DNA-PKcs,
ineachcase(Fig.1C).Asubstratehavingonly19bpofduplex Ku70/80,andArtemisisshown(Fig.1D).
DNA(48/19)sustained50%lesscleavage,andtheusual5-base DNA-PKcsandKuDependenceofArtemisActivityonLong3(cid:3)
overhang specificity was lost, with an 11-base overhang (30- Overhangs—Previous work with immunoconjugated Artemis
mer)beingthemostfrequentproduct(Fig.1C). immobilizedonagarosebeadssuggestedthatArtemisnuclease
Takentogether,thesedataindicatethatArtemisiscapableof cleavedlong3(cid:3)overhangstoalengthof(cid:5)5bases,inareaction
trimming 3(cid:3) overhangs at least as long as 25 bases, with the thatrequiredDNA-PKcsbutwasunexpectedlyindependentof
incisionsitebeingpredominantly5nucleotidesfromthedou- thepresenceorabsenceofKu(3).ToassesstheDNA-PKcsand
blestrand/singlestrandtransition.Asfootprintingstudiesindi- KudependenceofDNAcleavagebypurifiedsolubleArtemis,a
cate that Ku plus DNA-PKcs protects (cid:5)28 bp at a DNA end substratehaving23bpofduplexDNAanda13-base3(cid:3)-hydrox-
(18), the apparent requirement for at least (cid:5)20 bp of duplex yl-terminated3(cid:3)overhang(36/23-mer,asabovebutlabeledat
FEBRUARY9,2007•VOLUME282•NUMBER6 JOURNALOFBIOLOGICALCHEMISTRY 3549
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
end-labeled(Fig.2)andinternally
labeled (Fig. 1) substrates indi-
catesthattherewasnegligibledeg-
radationofthe5(cid:3)terminiofthese
substrates.
Although Ku was not strictly
requiredforcleavage,theaddition
of purified Ku70/80 stimulated
Artemis nuclease activity 4-fold,
in a DNA-PKcs- and ATP-
dependent manner (Fig. 2A, lanes
5–8). Previous studies, showing
Ku-independent Artemis activity,
included excess 35-bp blunt-
ended DNA in Artemis nuclease
reactions to ensure robust activa-
tion of DNA-PKcs and phospho-
rylation of Artemis (3). However,
in reactions containing soluble
recombinant Artemis, addition of
the same 35-bp duplex did not
stimulate cleavage, even in the
absence of Ku (Fig. 2A, compare
lanes7and9).Moreover,inreac-
tions containing Ku, the addition
of excess 35-bp duplex reduced
Artemis-mediated cleavage of the
radiolabeled substrate 3-fold (Fig.
2A, compare lanes 8 and 10; see
supplemental Fig. 2). A titration
of 35-bp duplex into Artemis
nuclease reactions containing Ku
clearly showed that there was no
stimulatory effect at any concen-
tration(Fig.2C).Thus,thelabeled
substrate,ataconcentrationof(cid:5)5
FIGURE2.StimulationofArtemis-mediatedDNAcleavagebyKuandinhibitionbyexcessDNAends.
A, reaction requirements. The end-labeled 36/23 substrate bearing a 13-base hydroxyl-terminated 3(cid:3) nM,appearstobesufficienttopro-
overhang(5nM)wasincubatedfor30mininthepresenceof13nMKu,35nMDNA-PKcs,90nMArtemis, mote activation of DNA-PK and
and/or0.5(cid:1)Munlabeled35-merduplex.B,reactiontimecourse.Thesame36/23substratewastreatedwith Artemis. Quantification of tripli-
35nMDNA-PKcs,13nMKu,and90nMArtemisforthetimesindicated.Insetatrightshowsa20-foldoverexpo-
sureoflane3.C,concentration-dependentinhibitionbyexcessDNAends.The36/23substratewastreatedfor cate experiments with 240 nM
30minwith65nMKu,55nMDNA-PKcs,and240nMArtemisinthepresenceof4,16,60,250,or1000nM35-mer Artemis revealed that Ku stimu-
ndMupAlertxe.mD,ise,f5fe5cntMofDKNuAa-nPdKcesx,caensds1D3NnAMe(n(cid:2)d)soorn6o5vneMrh((cid:2)an(cid:2)g)cKleua.vEarrgoer.bTahress3h6o/2w3Ss.uEb.ostfr3a–te5wdeatsetrrmeainteadtiownitshfr2o4m0 lated overall cleavage by (cid:5)2-fold
atleastthreeindependentexperiments.TherewasnodetectablecleavagebyeitherArtemisorDNA-PKalone. at 13 nM and 4-fold 65 nM (Fig.
E,titrationofoverhangcleavagewithlimitingArtemisinthepresenceof35nMDNA-PKcsandthepresence(F) 2D). Most notably, this stimula-
orabsence((cid:4))of65nMKu.F,titrationofcleavageofaplasmidsubstratewithlimitingArtemis.Aninternally
tory effect was almost completely
labeledplasmidsubstratebearingahydroxyl-terminated13-baseoverhang(1nM)wastreatedwiththeindi-
catedconcentrationsofArtemisinthepresenceof35nMDNA-PKcsandthepresence(F)orabsence((cid:4))of13 abolished by the presence of
nMKuandcutwithTaqI.G,titrationofcleavageofthesameplasmidsubstratewithlimitingDNA-PKcsby90nM excess DNA ends (Fig. 2D), thus
Artemisinthepresence(F)orabsence((cid:4))of13nMKu.ErrorbarsinE–GshowS.E.ofthreeindependent
experimentswhenlargerthanthesymbols. explainingthelackofstimulationby
Ku in previous work where excess
the5(cid:3)endratherthaninternally)wasreactedwithArtemisplus 35-bpduplexwasroutinelyaddedtoArtemisreactions(3).
DNA-PKcomponentproteins(Fig.2A).Asexpected,cleavage TitrationswithlimitingArtemisrevealedthatKustimulated
ofthissubstratewascompletelydependentonArtemis,DNA- ArtemisactivityoverawiderangeofArtemisconcentrations
PKcs,andATPandyieldeda5-baseoverhangasthepredomi- (Fig.2E).InthepresenceofKu,theconcentrationofArtemis
nantproduct(49%oftotalcleavageinFig.2A,lanes3and8).A requiredforagivenlevelofcleavagewas10-foldlowerthanin
timecourseshowedthatthecleavagepatternwasestablished itsabsence.
withinthefirstfewminutesofthereaction(Fig.2B),consist- To generate substrates more similar to DSBs produced in
entwithendo-ratherthanexonucleolyticcleavage.Inaddi- vivo(i.e.havingverylongduplexregionsandshortoverhangs),
tion, the similarity of the cleavage patterns between the a 3(cid:3)-resected plasmid with an 11-base 5(cid:3) overhang was pre-
3550 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER6•FEBRUARY9,2007
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
Ku-dependent Serial Cleavage of
3(cid:3)Overhangs—Toexamineinmore
detailtheeffectofKuonthekinetics
andspecificityofcleavage,thesame
plasmid substrate used above was
treated with Artemis in the pres-
ence or absence of Ku for various
times, and the percentage of
uncleaved substrate as well as of
individual cleavage products was
quantified(Fig.3).Quantificationof
uncleavedsubstrateasafunctionof
time reveals that the rate of Arte-
mis/DNA-PKcs-mediated cleavage
was three times faster in the pres-
enceofKuthaninitsabsence(Fig.
3A). As expected, bands corre-
spondingtothetrimmedoverhangs
wereonlydetectedwhentheinter-
nallylabeledDNAendwasreleased
as an oligomer by treatment with
TaqI, and nuclease activity was
ATP-, DNA-PKcs-, and Artemis-
dependent (Fig. 3B). Notably, the
additionofKualsomarkedlyaltered
thedigestionpatternforthe3(cid:3)over-
hang(Fig.3B).IntheabsenceofKu,
the predominant cleavage product
FIGURE3.Ku-dependenttwo-stepcleavageofa13-baseoverhangbyArtemis.Aninternallylabeledplas- wasa5-baseoverhang(40%oftotal
midbearingahydroxyl-terminated13-base3(cid:3)overhangwastreatedwith90nMArtemisinthepresenceof cleavage),withsignificantamounts
35nMDNA-PKcsand0or13nMKuandthencutwithTaqI.A,overallkineticsofcleavage.Lossofinitialsubstrate
wasmeasuredasafunctionoftimeinthepresence(F)orabsence((cid:4))ofKu.B, requirementsfortheArtemis- of2–4-and6-baseoverhangprod-
mediatedcleavagereaction.Sampleswereincubatedfor30min,andvariouscomponentswereomittedfrom ucts also being apparent. In con-
thereaction,asindicated.Numberstotherightindicateoverhanglength.C,timecourseandspecificityof
trast, a 3-base overhang product
cleavage.ThedistributionofreactionproductsasafunctionoftimeinthepresenceorabsenceofKuwas
determinedbygelelectrophoresis.D,kineticsforformationandlossof5-base(F)and3-base(f)overhang consistently dominated the prod-
productsinthepresenceofKu.E,kineticsforformationof5-base(E)and3-base((cid:4))overhangproductsinthe
uctsinreactionscontainingKu.
absenceofKu.F,titrationofcleavagespecificitybyvariousconcentrationsofKu.Abundanceof6-base(Œ),
5-base(F),and3-base(f)overhangproductswasdeterminedfollowinga30-minincubation.ErrorbarsinA,D, To investigate further this Ku-
E,andFshowS.E.ofthreeindependentexperiments.Curve-fittingofthesedatatoatwo-stepreactionindi- induced change in product distri-
catedk (cid:4)0.11min(cid:1)1andk (cid:4)0.09min(cid:1)1inthepresenceofKu,andk (cid:4)0.033min(cid:1)1andk (cid:5)0.005min(cid:1)1
1 2 1 2 bution, reaction products were
theabsenceofKu,wherek andk aretherateconstantsforformationofthe5-baseoverhangandforits
1 2
conversionto3-baseoverhang,respectively(seesupplementalFig.3). examined at various reaction
times(Fig.3C).Inthepresenceof
Ku, the proportion of 5-base over-
pared,towhichvarious5(cid:3)-labeledoligomerswereligated.Proc- hangdecreasedfrom56%oftotalcleavageat2mintoonly6%at
essingof3(cid:3)overhangingterminionthesesubstrateswasana- 60min,whereastheproportionof3-baseoverhangincreased
lyzedbycuttingthetreatedplasmidwithTaqIorAvaItorelease from 10 to 54%, becoming the dominant product by 60 min.
thelabeledoligomerfromtheend,asdescribedpreviously(19). Moreover,theappearanceofthe3-baseproductwasconcom-
Consistentwithdataobtainedusingtheoligomericsubstrates, itantwiththedecreaseinthe5-baseproduct(Fig.3D).These
assays with a plasmid bearing a 13-base overhang revealed a dataareconsistentwithatwo-stepreaction,whereinthe5-base
significantstimulatoryeffectofKuonArtemisnucleaseactivity productisthesubstrateforasecondarynucleasecleavageyield-
overarangeofArtemisconcentrations(Fig.2F),withamaxi- ingthe3-baseproduct.Notably,intheabsenceofKuthiscross-
mum stimulation of 6-fold when Artemis was limiting (1–10 overpatternwasnotapparent(Fig.3E),andthedistributionof
nM).TitrationswithlimitingDNA-PKcsrevealedthatKustim- productswasessentiallyinvariantoverthecourseofthereac-
ulationofArtemisisDNA-PKcsconcentration-dependentand tions. In addition, this same effect was observed in titrations
thatoptimalnucleaseactivityrequiredDNA-PKcsconcentra- withlimitingKu,namelytheappearanceofthe3-baseproduct
tionsthatapproachmolarequivalencewithArtemis(Fig.2G). accompaniedbythelossofthe5-baseproduct(aswellasthe
Takentogether,thesedataaremostconsistentwithKustimu- 6-base product) as Ku concentrations increased (Fig. 3F). A
latingArtemisactivitythroughenhancingassemblyorrecruit- moredetailedcurve-fittinganalysisindicatedthatthesekinet-
mentofArtemis/DNA-PKcstoDNAtermini,therebyincreas- icscouldbeaccuratelydescribedbyatwo-stepmodel,andthat
ingnucleaseactivity. although Ku accelerated the initial generation of the 5-base
FEBRUARY9,2007•VOLUME282•NUMBER6 JOURNALOFBIOLOGICALCHEMISTRY 3551
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
5-baseoverhangproductwasevidentonlyatearlytimepoints.
At later times, the abundance of both the 5-base and 4-base
overhangs diminished, with concomitant increases in the 3-
and 2-base overhangs (Fig. 4D). These results are consistent
withasecondarycleavagethatremovestwoadditionalbases,as
seenabovewiththe13-baseoverhangsubstrate.Theanalogous
hydroxyl-terminatedsubstrate(Fig.4B)alsoshowedatime-de-
pendentincreaseintherelativeabundanceofshorterreaction
products, albeit less apparent than with the 3(cid:3)-PG substrate.
This is likely because the initial cleavage reaction proceeded
moreslowly(seesupplementalFig.4).
Theinfluenceof3(cid:3)-PGonArtemisnucleasespecificitywas
muchmoreevidentonsubstratesetswith5-and4-baseover-
hangs(Fig.5).Fora5-base3(cid:3)-hydroxyloverhang,two-nucle-
otide trimming was dominant (78% of cleavage products at 5
min), and single-nucleotide trimming was (cid:6)5%, whereas for
the analogous PG-terminated substrate single-nucleotide
removal accounted for nearly half (48%) of the total initial
cleavage(Fig.5A).Thissingle-nucleotideremovalwas10-fold
greaterinthepresenceofKuthaninitsabsence,reflectingboth
anoverallincreaseincleavageandachangeincleavagespeci-
ficity.Theresulting4-baseoverhangreachedamaximumby10
minandappearedtodecreaseslightlybetween10and30min,
whereas shorter reaction products continued to accumulate.
Just as with the 13-base overhang, this shift from longer to
shorteroverhangsat10–30minwasseenonlyinthosereac-
tionscontainingKu,consistentwithKu-dependentsecondary
cleavageofaportionofthelongestinitialproduct;withoutKu,
the three cleavage products accumulated in parallel (Fig. 5A,
graphs).Atlongertimes,therewaslittlechangeintheoverall
cleavagepattern,althoughtracesofshorterfragments(*),indi-
catingtrimmingintoduplexDNA,begantoaccumulate(Fig.
5A,topright).Thesedatadonotdistinguishwhetherthislate
FIGURE4.Trimmingof6-base3(cid:1)overhangsbyArtemis.Internallylabeled trimmingwasendo-orexonucleolytic,butsubsequentexperi-
plasmidsubstratesbearing6-basehydroxyl-orPG-terminated3(cid:3)overhangs
mentswhereinablunt-endedsubstratewascleavedatthesame
weretreatedwithArtemisandthencutwithTaqI.Reactionconditionsarethe
sameasinFig.3.A,generalstructureofshort3(cid:3)overhangsubstratesshowing sites (see Fig. 7) indicated the same dependence on activated
restrictionsitesusedforanalysis.B,cleavageofa6-base3(cid:3)-hydroxylover- DNA-PK.
hang.C,cleavageofa6-base3(cid:3)-PGoverhang.Arrowsabovethesequence
indicateinitialcleavagesites.Numberstotherightindicateoverhanglength. A similar effect of PG termini on nuclease specificity was
D,abundanceof5-base(F),4-base(f),3-base(Œ),and2-base((cid:1))overhang apparentwithsubstrateshaving4-baseoverhangs(Fig.5B),i.e.
productswasquantitatedfollowingtreatmentofthe3(cid:3)-PGsubstrate.Error
almostexclusively(88%)2-basetrimmingforthehydroxyl-ter-
barsshowrangeofvaluesobtainedintwoindependentexperiments,when
largerthanthesymbols.SeesupplementalFig.4foradditionalkineticdata. minatedsubstrate,butapproximatelyequal1-baseand2-base
trimming for the PG-terminated substrate. Unlike the longer
overhang 3-fold, Ku increased the rate of its conversion to overhangs,thehydroxyl-andPG-terminated4-baseoverhang
3-baseoverhangby(cid:5)20-fold(supplementalFig.3). substrates did not show loss of longer reaction products or a
3(cid:3)-Phosphoglycolate Termini Alter Artemis Nuclease Speci- significantchangeinproductdistributionovertime,suggesting
ficity on Shorter Overhangs—3(cid:3)-PG blocking groups did not that the 2- and 3-base overhangs are poor substrates for sec-
alterArtemis/DNA-PKendonucleaseactivitywhenonlong3(cid:3) ondaryArtemis-mediatedcleavageeveninthepresenceofKu.
overhangsdistaltothecleavagesite(Fig.1).Todeterminethe Inasmuchastherewasalmostno3-baseoverhangproduced
effect of 3(cid:3)-PG closer to the site of nuclease activity, plasmid from the hydroxyl-terminated 4-base substrate, the 3-base
substrates with 3–6-base overhangs, bearing either 3(cid:3)-PG or overhang generated from the 5-base PG-terminated sub-
3(cid:3)-hydroxyltermini,wereconstructed(Fig.4A)andsubjected stratemusthavearisenby2-nucleotidetrimmingoftheini-
totreatmentwithArtemis/DNA-PKfor2–30min.Fora6-base tialsubstrate.
3(cid:3)-hydroxyl overhang substrate, little if any single-nucleotide A titration with limiting Artemis (Fig. 5C) revealed that
trimmingwasobserved;2-baseremovalwaspredominant(60% cleavageofthehydroxyl-terminated5-basesubstratebyArte-
ofcleavageproductsat5min),withsome3-and4-baseremoval mis/DNA-PK shows the same 2-base removal specificity, as
also being evident (Fig. 4B). In contrast, the 3(cid:3)-PG substrate wellasnearlycompleteKudependence,overarangeofArtemis
showed, in addition to these products, subtle but detectable concentrations.WhenArtemiswaslimiting(5.6nM),thiscleav-
removal of a single 3(cid:3)-PG nucleotide (Fig. 4C). However, this agewas17timesgreaterinthepresenceofKu(26(cid:7)2%,n(cid:4)4)
3552 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER6•FEBRUARY9,2007
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
FIGURE5.Ku-dependentsingle-nucleotidetrimmingofplasmidsubstratesbearingPG-terminated5-baseand4-base3(cid:1)overhangs.Reactioncondi-
tionsandprotocolarethesameasinFig.4exceptthatKuwasomittedfromsomereactionsasindicated.Numberstotherightofthegelindicateoverhang
length.A,specificityandtimecourseofArtemis-mediatedtrimmingof5-basehydroxyl-andPG-terminated3(cid:3)overhangs.Abundanceof4-base(F,E),3-base
(f,(cid:4))and2-base(Œ,(cid:8))overhangproductswasdeterminedfollowingtreatmentinthepresence(closedsymbols)orabsence(opensymbols)ofKu.Notetrace
accumulationofshorterfragments(*)at1–2hindicatingcleavageof2–4basesintotheduplexregionofDNA.B,specificityandtimecourseofArtemis-
mediatedtrimmingof4-basehydroxyl-orPG-terminated3(cid:3)overhangs.Abundanceof3-base(F)and2-base(f)overhangproductswasdetermined.C,Ku
dependenceoftwo-nucleotidetrimmingofthehydroxyl-terminated5-baseoverhang.ReactionconditionsarethesameasinA,exceptthattheconcentration
ofArtemiswasvaried,andKuwasomittedfromsomereactionsasindicated.ErrorbarsindicateS.E.ofthreeindependentexperimentswhenlargerthanthe
symbols.DatainCarefromasingleexperiment;however,theKudependenceofcleavageofthissubstratewasconfirmedinfourindependentexperiments.
than in its absence (1.5 (cid:7) 0.1%, n (cid:4) 3), and increasing the demonstratingthattheArtemisreactionproducthasa3(cid:3)-hy-
Artemis concentration more than 20-fold did not fully com- droxylratherthana3(cid:3)-PGterminus(Fig.6C,lane6).Moreover,
pensateforthelackofKu(Fig.5C). both the putative 14-mer and the resulting 15-mer precisely
Finally,whensubstrateswith3-baseoverhangsweretreated comigratedwithauthenticmarkersofthepredictedsequence.
withArtemis,therewasalmostnoprocessingofthehydroxyl- Asexpected,theinitial3(cid:3)-PGsubstratewasunaffectedbytreat-
terminated substrate, whereas the PG-terminated substrate ment with terminal transferase, confirming that it had a
was processed almost exclusively (85–90%) by single-nucleo- blocked3(cid:3)terminus(Fig.6C,lane5).AsexpectedforArtemis-
tideremoval,90%ofwhichwasKu-dependent(Fig.6,AandB). mediatedcleavage,thebandcorrespondingtothe2-baseover-
Ananalogousphosphotyrosyl-terminatedsubstrate,typicalof hang product was only seen when Artemis, DNA-PKcs, and
strandbreaksinducedbytopoisomeraseIinhibitors(20),was ATPwerepresentinthereactionandwhenthesubstratewas
likewise processed primarily by single-nucleotide removal, subsequentlycleavedwithAvaIorTaqI(Fig.6D).
albeit3-foldlessefficiently(Fig.6,AandB).Toverifytheter- Thus,Artemisnucleasetrimsshorthydroxyl-terminated3(cid:3)
minalstructureoftheputative2-baseoverhangproductgener- overhangspredominantlybyremovaloftwoterminalnucleo-
atedbyremovaloftheterminal3(cid:3)-PGnucleotide,thisproduct tides, whereas 3(cid:3)-PG termini (and to a lesser extent 3(cid:3)-phos-
was subsequently treated with terminal transferase plus photyrosyl)alterthisspecificity,promotingtrimmingofasin-
ddGTP, which produced the expected (cid:2)1 nucleotide shift, glenucleotide.Forunmodifiedsubstrates,theminimumlength
FEBRUARY9,2007•VOLUME282•NUMBER6 JOURNALOFBIOLOGICALCHEMISTRY 3553
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
D)inanArtemis-,DNA-PKcs-,and
ATP-dependentmanner.Theaddi-
tion of Ku stimulated this activity
5-foldbutdidnotmarkedlyalterthe
productprofile.Thus,thisprocess-
ing,althoughslowerandhavingdif-
ferentcleavagespecificitythanthat
of3(cid:3)overhangs,exhibitedthesame
protein and cofactor requirements.
Similar results (not shown) were
obtained with a PG-terminated
1-base3(cid:3)overhang.
To assess whether the unlabeled
5(cid:3)-terminal strand was still intact
followingtrimmingofthe3(cid:3)termi-
nus, similar experiments were per-
formed wherein the labeled oli-
gomer was released by treatment
withAvaI,whichrequiresadouble
strand substrate. AvaI yielded
essentiallythesameproductprofile
asTaqI,whichcancleavebothsin-
FIGURE6.Single-nucleotidetrimmingofPG-andphosphotyrosyl-terminated3-base3(cid:1)overhangs gle-anddouble-strandedDNA(Fig.
byArtemis.A,timecourseandcleavagespecificity.Plasmidsubstrateswiththestructuresindicated 7E).Thisresultimpliesthat,atleast
(pTyr (cid:4) phosphotyrosyl) were treated with Artemis plus DNA-PK as in Fig. 3 and then cut with AvaI.
Numberstotherightindicateoverhanglength.B,quantitationofsingle-nucleotidetrimmingofa3(cid:3)-PG- for the majority of trimmed mole-
(F,E),3(cid:3)-phosphotyrosyl-(f),or3(cid:3)-hydroxyl-terminated(Œ)overhanginthepresence(closedsymbols)or cules, both strands were still intact
absence(opensymbols)ofKu.Errorbars showS.E.ofthreeindependentexperimentswitheachsubstrate, attheAvaIsiteandthattrimmingof
wgehneenralatergdebrythAarntetmheis-smymedbioatlse.dCt,rtimermmiinngalotfraan3s(cid:3)fe-PraGs-ete-mrmeidniaatteedd3e-xbteansesioovneorhfathneg.3F(cid:3)o-hllyodwrionxgyltrteearmtmineunst the 3(cid:3) end was accompanied by
withArtemisandAvaIasinA,samplesweretreatedwithterminaltransferase(TdT)plusddGTP.Lanes either no 5(cid:3) 3 3(cid:3) exonucleolytic
markedMcontain5(cid:3)-end-labeled14-or15-basefragmentsoftheexpectedsequence(TCGAGGAACGC-
resection or trimming of at most a
GACandTCGAGGAACGCGACG,respectively).Forthe3(cid:3)-PGsubstrate,essentiallyidenticalresultswere
obtainedwhenTaqIwasusedinsteadofAvaI(datanotshown).D,requirementsforsingle-nucleotide few ((cid:6)10) nucleotides from the 5(cid:3)
trimming.ThePG-terminated3-baseoverhangwastreatedwithArtemisplusDNA-PKfor30minasinA, terminus. Thus, Artemis plus
butvariousreactioncomponentswereomittedasindicated.
DNA-PK is clearly able to process
blunt3(cid:3)-PGends,albeitmoreslowly
ofoverhangthatwillsupportefficientArtemis-mediatedcleav- than short overhangs, with removal of a few adjacent
ageis4bases. nucleotides.
SlowTrimmingof3(cid:3)-PG-terminatedBluntEnds—Tofurther Toxicityof3(cid:3)-Phosphoglycolate-terminatedDSBsinArtemis-
investigatethespecificityofArtemis/DNA-PK,aseriesofplas- deficientCells—TheabovedataclearlyshowthatArtemiscan
midsubstratesbearinga3(cid:3)-PGterminusona2-baseoverhang, process3(cid:3)-PG-terminatedDSBs.However,althoughhypersen-
a blunt end, or a 2-base recessed 3(cid:3) end were generated and sitivityofArtemis-deficientmousefibroblaststobleomycinhas
assayed for susceptibility to Artemis nuclease (Fig. 7A). been reported (21), a role for Artemis in processing of PG-
Althoughallsubstratesweresubjecttosomedegreeofdiges- terminated DSBs in intact human cells has not been estab-
tion, there was a pronounced decrease in nuclease efficiency lished.Toaddressthisquestion,weassayedthetoxicityoftwo
andspecificityforPG-terminatedsubstrateshavingoverhangs drugsthatinducedifferenttypesof3(cid:3)-PG-terminatedDSBsin
oflessthan3bases.Forexample,a30-minArtemistreatmentof vivo.BleomycintreatmentgivesrisetoDSBs,nearlyallofwhich
asubstratewithaPG-terminatedbluntendor2-base3(cid:3)over- have either blunt ends or single-base 5(cid:3) overhangs, with
hangyieldedsmallamountsofmultiplecleavageproducts,each 5(cid:3)-phosphateand3(cid:3)-PGterminiatbothendsofthebreak.Neo-
ofwhichaccountedfor(cid:6)10%oftheinitialsubstrate(Fig.7,A carzinostatin-induced DSBs have at one end a 5(cid:3)-phosphate
and B). To assess the requirements, specificity, and extent of anda3(cid:3)-phosphateona2-base3(cid:3)overhang;theoppositeend
this processing, nuclease assays with the 3(cid:3)-PG blunt-ended hasa5(cid:3)-aldehydeandeithera3(cid:3)-PG((cid:5)75%)ora3(cid:3)-phosphate
plasmid substrate were carried out for up to 2 h (Fig. 7B). ((cid:5)25%)ona1-base3(cid:3)overhang(14,15).Followingtreatment
Although control reactions with Ku and/or DNA-PKcs alone ofexponentiallygrowingcellswitheachoftheseagents,bro-
showed no significant trimming, those containing Artemis modeoxyuridine incorporation into (cid:5)10,000 cells was meas-
indicatedtime-dependenttrimmingcorrespondingtoremoval ured(cid:5)24haftertreatment,andfromthesedatatheproliferat-
of2–4basesfromthe3(cid:3)-PG-terminatedbluntends(Fig.7B). ingfractionofcellswascalculated(Fig.8).
Although this trimming proceeded about seven times more We previously showed that fibroblasts from Artemis-defi-
slowlythantrimmingofa5-base3(cid:3)overhang,by2hnearlyhalf cientSCIDApatientsexhibitsignificantlyelevatedsensitivityto
(43(cid:7)3%,n(cid:4)3)ofthebluntendsweretrimmed(Fig.7,Cand x-irradiationbutamuchlessersensitivitytoDSBsinducedby
3554 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER6•FEBRUARY9,2007
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
treatmentwithx-rays,neocarzinos-
tatin, and bleomycin, all of which
induce 3(cid:3)-PG-terminated DSBs,
results in at least 2-fold greater
accumulation in G /M in cycling
2
SCIDA cells than in normal cells
(Fig.8B).Comparisonoftherelative
toxicities of these three agents in
multiple experiments reveals that
the hypersensitivity of SCIDA cells
is about the same for x-rays and
neocarzinostatin but less for bleo-
mycin (Table 1). Together, these
datasuggestthatx-raysandradiom-
imetic drugs induce DSBs or other
lesionsthatrequireArtemisforres-
olution prior to continued cellular
proliferation.
DISCUSSION
Datapresentedhereshowthatin
the context of a variety of model
DSB substrates, purified histidine-
tagged Artemis efficiently cleaves
long3(cid:3)overhangstoalengthof4–5
nucleotides,inareactiondependent
on ATP and catalytically active
DNA-PKcs.Ourreactionswithsol-
uble purified proteins also reveal
significant stimulation of Artemis
activity by Ku for all substrates
tested (Figs. 2, 3, and 5–7). Inas-
muchasKuisnormallyrequiredfor
efficient DNA-PK assembly and
self-activationonDNAends,except
at very low ionic strength (22, 23),
and catalytically active DNA-PK is
requiredtoactivateArtemisnucle-
ase, it is expected that Ku would
have a stimulatory effect on Arte-
mis-mediated DNA cleavage. The
reported Ku independence for
FIGURE7.Artemis-mediatedtrimmingof3(cid:1)-PGbluntendsandcomparisonwithother3(cid:1)-PGsubstrates. immobilized Artemis activity (3) is
ReactionconditionsarethesameasinFig.3.A,indicated3(cid:3)-PG(F)substratesweretreatedwithArtemisplus likely because of the presence of
DNA-PKfor30minandcutwithAvaI.B,bluntend3(cid:3)-PGsubstratewastreatedwithArtemisplusDNA-PKcsfor
excessDNAendsinthosereactions,
theindicatedtimesinthepresenceorabsenceofKuandthencutwithTaqI.C,timecourseforoverallcleavage
ofthebluntand5-baseoverhang3(cid:3)-PGsubstrates.Thefractionofunprocessedsubstratewascalculated. which in our hands almost com-
D,timecourseandspecificityofproductformationfortheblunt3(cid:3)-PGsubstrate.Abundanceofproducts pletely suppresses the stimulatory
correspondingtotheremovalof2(F),3(f),or4(Œ)baseswasquantitatedfollowingtreatmentofthe
blunt-ended3(cid:3)-PGsubstratewithArtemisplusDNA-PK.Errorbarsshowmeans(cid:7)S.E.ofthreeindependent effectofKu(Fig.2).
experimentswhenlargerthanthesymbols.E,sameasB,exceptthatfollowingincubationwithArtemis,halfof AtlowDNAterminusconcentra-
eachsamplewastreatedwithAvaIandhalfwastreatedwithTaqI. tions (1–5 nM), a Ku-mediated
recruitmentofArtemis/DNA-PKcs
etoposide (6). Here we find that human telomerase reverse toDNAendsshouldmarkedlyimprovethereactionrate,asis
transcriptase-immortalizedSCIDAfibroblastsshowhypersen- observed (Fig. 2D; Fig. 3, A and C). At high DNA terminus
sitivitytotheDSB-inducingagentbleomycin,albeitnotasgreat concentrations(1(cid:1)M),theeffectofanyincreaseinaffinitywill
astheirsensitivitytox-rays(Fig.8A).Interestingly,DSBspro- beatleastpartiallyabrogatedbyincreasedbindingtounlabeled
ducedbyneocarzinostatin((cid:5)75%ofwhichbearanoverhang- competitorDNA,andtheincreaseincleavagewillbelesspro-
ing3(cid:3)-PGterminusatoneendofthebreak)appeartobenearly nounced, as seen in Fig. 2. The finding that unlabeled 35-bp
astoxictoSCIDAcellsasx-rays(Fig.8AandTable1).Similarly, duplexdoesnotcompetewithlabeledsubstrateintheabsence
FEBRUARY9,2007•VOLUME282•NUMBER6 JOURNALOFBIOLOGICALCHEMISTRY 3555
ArtemisProcessingof3(cid:1)-PhosphoglycolateTermini
Artemisunderthesameconditions
resulted almost exclusively in
2-base trimming to a 3-base over-
hang,inareactionthatwasalmost
completely dependent on Ku (Fig.
5C).Othersubstrates(Figs.4Cand
5A)show,tovaryingdegrees,simi-
lar progression from longer to
shorter cleavage products with
time,andalthoughtime-dependent
changesinArtemisspecificitycan-
notbeformallyruledout,allofthe
data can be explained by the same
two-stepmechanismwhereinapor-
tionoftheinitialcleavageproducts
are subsequently trimmed, 2 bases
atatime,untiltheoverhanglength
isreducedto3basesorless.
Although3(cid:3)-blockingPGmoieties
onlong3(cid:3)overhangshavenodiscern-
ibleinfluenceonArtemisendonucle-
ase activity, the presence of 3(cid:3) PG
closer to the site of cleavage signifi-
cantlyaltersArtemisnucleasespec-
ificity,promotingsingle-nucleotide
removal from short overhangs.
FIGURE8.CellularproliferationassaysshowinghypersensitivityofSCIDAcellstox-rays,neocarzinosta- Most strikingly, Artemis trims a
tin,andbleomycin.A,percentofproliferating(BrdUrd-positive)cells29haftertreatmentwitheachagentwas
determinedandnormalizedtotheuntreatedcontrol.Shownabovethetoxicitydataarethestructuresof 3-base 3(cid:3)-PG overhang almost
typicalbleomycinandneocarzinostatin-inducedDSBs,insomecasesbearing3(cid:3)-PG(F)or5(cid:3)-aldehyde(f) exclusively by single-nucleotide
termini.Anestimated75%ofneocarzinostatin-inducedDSBshavethestructureshown,whereastheremain-
removal,whileleavingananalogous
derhavea3(cid:3)-phosphateratherthanan3(cid:3)-phosphoglycolate.Radiation-inducedDSBswillhaveshort5(cid:3)or3(cid:3)
overhangsofvariedlengthwithpredominantly5(cid:3)-phosphateterminiand(cid:5)50%3(cid:3)-PGtermini.B,cellcycle 3(cid:3)-hydroxyl substrate largely intact
distributionofBrdUrd-labeledcells29haftertreatmentwith5gray(Gy)x-rays,10nMneocarzinostatin,or (Fig.6).
8(cid:1)g/mlbleomycin.
Basedontheseandotherresults
(Figs.1–7),thesiteofArtemis-me-
ofKu,butisaneffectivecompetitorinthepresenceofKu(Fig. diatedcleavageof3(cid:3)overhangsappearstobedependentonthe
2A), also suggests that the Artemis-DNA-PK complex has following three criteria: 1) a requirement for either 2 nucleo-
higheraffinityforbluntendsinthepresenceofKuthaninits tidesoranucleotideplusa3(cid:3)-PG3(cid:3)tothecleavagesite;2)a
absence. Conversely, the finding that cleavage of very short requirementforatleast2unpairednucleotides5(cid:3)tothecleav-
overhangsismorestronglydependentonKuthaniscleavageof age site; and 3) a preference for cleavage 4–5 bases from the
longeroverhangs(compareFigs.2Fand5C)isconsistentwitha singlestrand/doublestrandtransition.Itislikelythatforlong
model wherein contacts between Artemis and the long over- overhangs,DNA-PKatthedoublestrand/singlestrandtransi-
hangcanenhanceaffinityandtherebypartiallycompensatefor tion positions the Artemis active site for cleavage 4–5 bases
thelackofKu.ItisalsonotablethatexcessDNAends,which fromthetransition.However,onshorteroverhangsthisposi-
shouldstimulateglobalDNA-PKkinaseactivity,donotstimu- tioningispreemptedbythemorestringentrequirementfor2
lateArtemisnucleaseactivity(Fig.2,A,C,andD).Rather,opti- nucleotides3(cid:3)tothecleavagesite,thusshiftingthecleavagesite
malnucleaseactivityisseenwiththelowestconcentrationsof closer to the double strand/single strand transition. A 3(cid:3)-PG
DNAends,consistentwithamodelinwhichKuandautophos- canapparentlypartiallysubstitutefortheterminalnucleotide,
phorylatedDNA-PKcsformastablecomplexwithlong3(cid:3)over- resulting in single-nucleotide removal from short PG-termi-
hangs(24),sothatonlylocalactivationofDNA-PKonagiven natedoverhangs.Unmodifiedoverhangsshorterthan4bases,
DNAendisimportantforArtemisnucleaseactivation. andPG-terminatedoverhangsshorterthan3bases,aremuch
In the case of a long (13-base) overhang on a plasmid sub- lessefficientlyprocessed,likelyduetothefactthattherequire-
strate,thepresenceofKunotonlystimulatedthecleavagereac- mentsfor2bases3(cid:3)and2unpairedbases5(cid:3)tothelesioncannot
tionbutalsomarkedlyalteredtheproductdistribution(Fig.3). besimultaneouslysatisfied.Nevertheless,veryshortoverhangs,
Thedataaremostconsistentwithatwo-stepprocess,wherein bluntends,andevenrecessedendsbearing3(cid:3)-PGterminishow
theinitialcleavageremovestheextended3(cid:3)singlestrandtail, significant Artemis-mediated processing (Fig. 7), albeit at a
leavinga5-baseoverhangwhich,onlyinthepresenceofKu,is much slower rate than longer overhangs, and after 2 h the
subsequentlytrimmedbyanadditional2bases.Consistentwith 5-base overhang also shows traces of trimming into duplex
thisidea,directtreatmentofa5-baseoverhangsubstratewith regions.Overall,theobservedpatternsofcleavageareconsist-
3556 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER6•FEBRUARY9,2007