Unifying the DNA End-processing Roles of the Artemis Nuclease

16 Pages·2015·3.55 MB·English

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THEJOURNALOFBIOLOGICALCHEMISTRY VOL.290,NO.40,pp.24036–24050,October2,2015
©2015byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Unifying the DNA End-processing Roles of the Artemis
Nuclease
KU-DEPENDENTARTEMISRESECTIONATBLUNTDNAENDS*
Receivedforpublication,July24,2015,andinrevisedform,August12,2015 Published,JBCPapersinPress,August14,2015,DOI10.1074/jbc.M115.680900
HowardH.Y.Chang,GoWatanabe,andMichaelR.Lieber1
FromtheDepartmentsofPathology,BiochemistryandMolecularBiology,BiologicalSciences,andMolecularMicrobiologyand
Immunology,UniversityofSouthernCaliforniaKeckSchoolofMedicine,NorrisComprehensiveCancerCenter,
LosAngeles,California
Background:ArtemisisanucleasethatisnecessaryforhairpinopeninginV(D)Jrecombination.
Results:ArtemisactiononbluntDNAendsisdependentonDNAsequence(breathing)andKu.
Conclusion:BreathingofbluntDNAendsintoatransientss/dsDNAboundaryisneededforArtemisaction.
Significance:UnificationofArtemisnucleaseactionexplainsthefeaturesofNHEJandV(D)Jrecombination.
Artemisisamemberofthemetallo-(cid:2)-lactamaseproteinfam- radicals,replicationerrors,andinadvertentcleavagebynuclear
ily of nucleases. It is essential in vertebrates because, during enzymes(4).Manyofthesepathologicalbreaksandthephysi-
V(D)J recombination, the RAG complex generates hairpins ologicalbreaksinV(D)Jrecombinationrequirethenonhomo-
whenitcreatesthedoublestrandbreaksatV,D,andJsegments, logousDNAend-joining(NHEJ)nucleaseArtemistorepairthe
andArtemisisrequiredtoopenthehairpinssothattheycanbe DNA. In NHEJ, the DSB is first recognized by the Ku het-
joined.Artemisisadiverseendo-andexonuclease,andcreating erodimer(Ku70/80),whichactsasa“toolbelt”towhichother
a unified model for its wide range of nuclease properties has NHEJproteinsarerecruitedasneeded.WhenDNAresectionis
beenchallenging.HereweshowthatArtemisresectsiteratively required, a DNA-dependent protein kinase catalytic subunit
into blunt DNA ends with an efficiency that reflects the AT- (DNA-PKcs)isrecruitedincomplexwithArtemis.DNA-PKcs
richnessoftheDNAend.GC-richendsarenotcutbyArtemis undergoes autophosphorylation and activates Artemis endo-
alone because of a requirement for DNA end breathing (and nucleaseactivity(5,6).ActivatedArtemisthengainstheability
confirmedusingfixedpseudo-Ystructures).AllDNAendsare tocutmanyDNAsubstratesatthesingletodoublestrandDNA
cutwhenboththeDNA-dependentproteinkinasecatalyticsub- boundaries(7)(Fig.1).
unitandKuaccompanyArtemisbutnotwhenKuisomitted. Artemis is in the metallo-(cid:2)-lactamase family of nucleases,
ThesearethefirstbiochemicaldatademonstratingaKudepen- characterizedbyconservedmetallo-(cid:2)-lactamaseand(cid:2)-CASP
denceofArtemisactiononDNAendsofanyconfiguration.The domains.ThisfamilyofnucleasesisabletohydrolyzeDNAor
actionofArtemisatbluntDNAendsisslowerthanatoverhangs, RNAinvariousconfigurations(8).Artemisalonehasintrinsic
consistent with a requirement for a slow DNA end breathing 5(cid:2) exonuclease activity on single-stranded DNA (ssDNA) (9).
stepprecedingthecut.TheATsequencedependence,theorder On duplex DNA, Artemis, in complex with DNA-PKcs, has
ofstrandcutting,thelengthofthecuts,andtheKu-dependence endonucleaseactivityonthe5(cid:2)and3(cid:2)DNAoverhangsthatare
ofArtemisactionatbluntendscanbereconciledwiththeother oftencreatedbypathologicalDNAbreaksandonDNAhair-
nucleolyticpropertiesofbothArtemisandArtemis(cid:2)DNA-PKcs pins that are formed during V(D)J recombination. Therefore,
inamodelincorporatingDNAendbreathingofbluntendsto patientslackingArtemissufferfromseverecombinedimmu-
form transient single to double strand boundaries that have nodeficiencybecauseofadefectinantibodyformation(10).
structuralsimilaritiestohairpinsandfixed5(cid:3)and3(cid:3)overhangs. WehavedeterminedbiochemicallythatArtemis,incomplex
with DNA-PKcs, resects 5(cid:2) and 3(cid:2) DNA overhangs to create
DNA end structures that are able to be ligated by the DNA
Individingmammaliancells,thereareanestimated10DNA ligase IV-XRCC4 complex (11, 12). At 5(cid:2) overhangs, Artemis
double-strandedbreaks(DSBs)2perdaypercell(1–3).These cutsdirectlyatthess/dsDNAboundary.However,whenpro-
pathological DSBs arise from ionizing radiation, oxygen free cessing3(cid:2)overhangsandDNAhairpins,itpreferentiallyleaves
a4-ntoverhang(7).PerfectDNAhairpinshavestericallycon-
strainedtightturnsand,therefore,havess/dsboundariesatthe
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth
(toM.R.L.).Theauthorsdeclarethattheyhavenoconflictsofinterestwith lasttwobasepairs(4nt)(13).Thismaymakethemstructurally
thecontentsofthisarticle. similar to DNA overhangs. From these observations, we pro-
1Towhomcorrespondenceshouldbeaddressed:Tel.:[phone];E-mail: posed that Artemis activity on duplex DNA can be unified
[email protected].
2Theabbreviationsusedare:DSB,doublestrandbreak;NHEJ,nonhomo- underamodelinwhichArtemis(cid:2)DNA-PKcsbindstothess/ds-
logousDNAend-joining;(cid:2)-CASP,namedafterfourrepresentativemetallo- DNAboundarytooccupy4ntalongthesingle-strandedseg-
(cid:2)-lactamases:CPSF,Artemis,SNM1,PSO2;CPSF,cleavageandpolyadenyl- ment.Thisbindingisfollowedbypreferentiallynickingthe3(cid:2)
ationspecificityfactor;SNM,senstivetonitrogenmustard;PSO,psoralen;
sideofthose4nt.Althoughthecurrentmodelisextensive,it
RAG,recombinationactivatinggene;DNA-PKcs,DNA-dependentprotein
kinasecatalyticsubunit;ss,single-stranded;ds,doublestranded. lacks information on whether Artemis can cut blunt-ended
This is an open access article under the CC BY license.
24036 JOURNALOFBIOLOGICALCHEMISTRY VOLUME290•NUMBER40•OCTOBER2,2015
ArtemisandKuatDNAEnds
DNAthatcanbegeneratedbychemotherapeuticagents,free ACATCCGTCAAGTAAGATGCAGACACTCCCC-3(cid:2);
radicals,orionizingradiation(14). HC84,5(cid:2)-biotin-AAAACATCCGTCAAGTAAGATGCA
HereweexploretheactionofArtemisatbluntDNAends, GAC ACT GGG G-3(cid:2); HC85, 5(cid:2)-TTT TTT TTT TTT TAG
whichappearssubstantiallyweakerthanitsactionatss/dsDNA TGTCTGCATCTTACTTGACGGATGTTTT-3(cid:2);HC86,
boundaries.Wefoundthatresectionatbluntenddependson 5(cid:2)-TTT TAG TGT CTG CAT CTT ACT TGA CGG ATG
theDNAsequenceoftheduplexend,withAT-richDNAends AAA-3(cid:2);HC87,5(cid:2)-TTTTAGTGTCTGCATCTTACTTGA
being resected by Artemis alone but GC ends hardly at all. CGGATGGGG-3(cid:2);HC88,5(cid:2)-TTTTAGTGTCTGCATCTT
WhenArtemisisaccompaniedbyDNA-PKcs,itcanactatall ACTTGACGGATGCCC-3(cid:2);HC89,5(cid:2)-phosphate-TTTTCA
blunt ends, but in a Ku-dependent manner. This is the first TCCGTCAAGTAAGATGCAGACACTAAAA-biotin-3(cid:2);
documentationshowingthatKucanmodulateArtemisactiv- HC90, 5(cid:2)-phosphate-CCC CCA TCC GTC AAG TAA GAT
ity. The sequence dependence and other features of Artemis GCAGACACTAAAA-biotin-3(cid:2);HC91,5(cid:2)-phosphate-GGG
action permit the unification of all nucleolytic properties of GCATCCGTCAAGTAAGATGCAGACACTAAAA-bi-
Artemis into a single model for its action at ss/dsDNA otin-3(cid:2);HC98,5(cid:2)-CCCCCATCCGTCAAGTAAGATGCA
boundaries. GAC ACT AAA A-biotin-3(cid:2); and HC99, 5(cid:2)-GGC CAG TGT
CTGCATCTTACTTGACGGATGTTTT-3(cid:2).
ExperimentalProcedures
ProteinExpressionandPurification—Wild-typeArtemisand
Oligonucleotides and DNA Substrates—The Oligonucleo- catalytically inactive ArtemisH115A (ARM14) were purified as
tides used in this study were synthesized by Integrated DNA describedpreviously(9,15).Briefly,Sf9insectcells(LifeTech-
Technologies,Inc.(SanDiego,CA).Theoligonucleotideswere nologies,catalogno.11496-015)wereinfectedwithbaculovirus
purifiedusingdenaturingPAGE,andtheconcentrationswere containing C-terminal His-tagged Artemis DNA. Cells were
determined spectrophotometrically. 5(cid:2) end labeling of DNA lysedandpurifiedbynickel-nitrilotriaceticacidaffinitychro-
substrates was done with [(cid:3)-32P]ATP (3000 Ci/mmol) matography,anionexchangechromatography,andsizeexclu-
(PerkinElmer Life Sciences) and T4 polynucleotide kinase sionchromatography.TheKu70-Ku80complexwaspurifiedas
(PNK)(NewEnglandBiolabs)accordingtotheinstructionsof describedpreviously(11).Briefly,Hi-5insectcells(Invitrogen,
themanufacturer.Unincorporatedradioisotopewasremoved catalogno.B855-02)werecoinfectedwithbaculovirusescon-
by using Sephadex G-25 spin columns (Enzymax). To create tainingtheDNAforC-terminalHis-taggedKu70anduntagged
dsDNA,theunlabeledcomplementarystrandwasadded,and Ku80. Cells were lysed and purified by nickel-nitrilotriacetic
thesamplewasboiledfor5mintoinactivateT4PNK.TheDNA acidaffinitychromatography,dsDNA(oligo)affinitychroma-
wasallowedtoslowlycooltoroomtemperatureandthenincu- tography,andanionexchangechromatography.Thepurifica-
batedovernightat4°Ctopromoteproperbasepairingofthe tion of endogenous DNA-PKcs from HeLa cells has been
complementarystrand. describedpreviously(16).KuandDNA-PKcswereconfirmed
3(cid:2) end labeling was performed by fill-in synthesis of tohavenodetectablenucleaseactivities.Atleastthreeentirely
[(cid:4)-32P]dNTP(3000Ci/mmol)(PerkinElmerLifeSciences)onto independent fresh preparations of Artemis and DNA-PKcs
preannealeddsDNAwitha1-nt5(cid:2)overhangwiththeKlenow wereusedinthesubsequentassays,andthesepreparationsgen-
fragmentofDNApolymeraseI(3(cid:2)35(cid:2)exo(cid:3))(NewEngland eratedindistinguishableresultsfromoneanother.
Biolabs) to create a blunt-ended substrate. Unincorporated Biochemical Nuclease Assay—The in vitro DNA nuclease
radioisotopewasremovedasstatedabove. assayswereperformedinavolumeof10(cid:5)lwithabuffercom-
Thesequencesoftheoligonucleotidesusedinthisstudywere positionof25mMTris-HCl(pH8.0),75mMKCl,10mMMgCl ,
2
as follows: HC33, 5(cid:2)-GCG GAG TGT CTG CAT CTT ACT and1mMDTT.Thereactionsconsistedof20nMof32P-labeled
TGACGGATGCAATCGTCACGTGCTAGACTACTG DNAincubatedwith0.5mMATP(or0.5mMAMP-PNP),50nM
GTC AAG CGG ATC TTA GGG G-3(cid:2); HC34, 5(cid:2)-CCC CTA Ku,50nMDNA-PKcs,and50nMArtemis(or50nMARM14)at
AGATCCGCTTGACCAGTAGTCTAGCACGTGACG 37°C for 30 min unless specified otherwise. The time course
ATTGCATCCGTCAAGTAAGATGCAGACACTCCG assayswereperformedinavolumeof12or15(cid:5)l.Theladders
C-3(cid:2); HC57, 5(cid:2)-TTT TAG TGT CTG CAT CTT ACT TGA werecreatedbyincubating40nM32P-labeledssDNAwith0.5
CGG ATG TTT T-3(cid:2); HC58, 5(cid:2)-AAA ACA TCC GTC AAG milliunits/ml snake venom phosphodiesterase I (Sigma-Al-
TAAGATGCAGACACTAAAA-3(cid:2);HC73,5(cid:2)-phosphate- drich,catalogno.P3243)for15minat37°C.Allreactionswere
AAAACATCCGTCAAGTAAGATGCAGACACTAAA stoppedbytheadditionofanequalvolumeof98%formamide,
A-biotin-3(cid:2); HC74, 5(cid:2)-TTT TAG TGT CTG CAT CTT ACT heatedto95°Cfor5min,placedonicefor5min,andanalyzed
TGA CGG ATG TTT-3(cid:2); HC76, 5(cid:2)-biotin-AAA ACA TCC ona12or14%denaturingPAGE.Thegelswerethendriedand
GTC AAG TAA GAT GCA GAC ACT AAA A-3(cid:2); HC77, exposed to a phosphorimaging screen overnight. The screen
5(cid:2)-phosphate-A*A*A*A*C*A*T*C*C*G*TCAAGTAAGAT was scanned, and quantification was performed in Quantity
GCA GAC ACT AAA A-biotin-3(cid:2) (the asterisks represent One(cid:4)(Bio-Rad).
phosphorothioate bonds); HC79, 5(cid:2)-AAA AAG TGT CTG
CAT CTT ACT TGA CGG ATG TTT T-3(cid:2); HC80, 5(cid:2)-GGG Results
GAGTGTCTGCATCTTACTTGACGGATGTTTT-3(cid:2); ArtemisCanResectBluntDNAEndsButataMuchSlower
HC81, 5(cid:2)-CCC CAG TGT CTG CAT CTT ACT TGA CGG RateThanatDNAOverhangs—InFig.1,weshowasummaryof
ATGTTTT-3(cid:2);HC82,5(cid:2)-biotin-AAAACATCCGTCAAG major Artemis substrates and preferred positions of Artemis
TAAGATGCAGACACTTTTT-3(cid:2);HC83,5(cid:2)-biotin-AAA cuttingonthosesubstrates.Theopenthickarrowsindicatethe
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ArtemisandKuatDNAEnds
FIGURE1.OptimalsubstratesforArtemishavess/dsDNAboundariesthatdirectArtemistothepointofpreferrednucleaseaction.Shownarethe
knownmajorsubstratesforArtemis(7,11).TheputativeorientationandpositionoftheArtemisenzymeareindicatedbytheopenthickarrows,consistentwith
thepreferenceofArtemisforss/dsDNAboundariesanditspropensitytocutonthe3(cid:2)sideofan(cid:4)4-ntssDNAregionatthisboundary.Thepreferredcutting
sitesofArtemisarenotedwithredarrows.WeproposethatweakeractivitymaybeseenonvariantsofthesestructureswhenconditionsfavorDNAbreathing
intooneoftheformsshownhere.Inadditiontothesubstratesshown,Artemishas5(cid:2)exonucleaseactivityat5(cid:2)protrudingDNAends(9),whichistheonly
activitythatdoesnotrelyonDNA-PKcs(underMg2(cid:5)conditions)(seetext).ArtemisalsohasveryweakendonucleaseactivityonssDNA,whichisstimulatedby
DNA-PKcsandcanbeslightlystrongerforssDNAthatcanself-annealorfoldbackatinternalpositionsbecauseofself-complementarity(6).
putativeorientationandpositionoftheArtemisenzyme,and resultinginamoreintenseband.Incontrast,the34-bpblunt-
theredarrowsindicatethepreferredcuttingsites.Artemisis endedsubstrateproducedadistributionofcuts1–6ntintothe
knowntohaveendonucleaseactivityonDNAhairpinsandon duplexDNAend(Fig.2A,lanes7–12).
5(cid:2) and 3(cid:2) overhangs of duplex DNA, which is dependent on Thecutproductswerethenquantifiedasapercentageofthe
DNA-PKcsactivity(Fig.1,toprow).Previousbiochemicaldata totalsubstrateandplottedasafunctionoftime(Fig.2B).The
haveindicatedthatArtemispreferentiallycutsonthe3(cid:2)sideof rate of activity was determined by calculating the slope from
a4-ntssDNAsegmentdirectlyadjacenttoass/dsDNAbound- theaverageoftwoentirelyindependentexperiments(product
ary(7,11).TheotherknownArtemissubstrateslistedinFig.1 percentperminuteofreaction).(Severaladditionalverysimilar
(center and bottom rows) are physiologically relevant because experiments further supported these observations.) The 5(cid:2)
theymayarisefromreplicationerrorsorbyinadvertentaction overhang substrate was processed (cid:4)3-fold faster than blunt-
by nuclear enzymes. Nevertheless, we found that Artemis is endedDNAand2-foldfasterthanssDNA(Fig.2C).Althoughit
abletocutthesesubstratesonthessDNAportionnearestthe isknownthatArtemishasendonucleaseactivityonssDNA(6),
ss/dsDNAboundaries.Wethenwonderedwhatwouldoccurat it was surprising to observe that the activity was only 2-fold
bluntDNAendswhensuchendsmaybeablebreatheopento slowerthanatoverhangs.UnlikehomopolymericssDNAsub-
formatransientpseudo-Ystructure(Fig.1,centerrowandcen- strates, we suspect that the ssDNA substrate used here may
tercolumn). haveself-annealedtoproducepartialduplexDNAstructures.
WeinitiallyperformedatimecourseexperimentwithArte- ArtemisEndonucleaseActivityonBluntDNAEndsIsStrictly
mis and DNA-PKcs on 9-nt 5(cid:2) overhang, blunt-ended, and DependentonDNA-PKcsandATPandIsModulatedbyKu—
ssDNAsubstrates(Fig.2).Predictably,thepredominantcuton Asmentioned,Artemishasintrinsic5(cid:2)exonucleaseactivityand
the 5(cid:2) overhang substrate is at 9 nt, producing a blunt-ended DNA-PKcs-dependent endonuclease activity. Therefore, we
product (Fig. 2A, lanes 1–6). The 34-nt ssDNA substrate wondered whether this activity also applied to blunt-ended
(whichmatchedthesequenceofthetopstrandoftheduplex DNA.Weincubateda5(cid:2)-radiolabeled34-bpdsDNAsubstrate
portionoftheothersubstrates)producedrandomendonucleo- flanked with dTs in a reaction with Ku, DNA-PKcs, Artemis,
lytic cuts (Fig. 2A, lanes 13–18). In addition, the apparent andARM14(catalyticallyinactiveArtemisH115A)withorwith-
intense band at (cid:4)18 nt may be explained by the palindromic outATPfor30minat37°(Fig.3).IntrinsicArtemis5(cid:2)exonu-
5(cid:2)-TGCA-3(cid:2)onthe11th-14thnt,allowingthesubstratetoself- clease activity was able to resect the first 5(cid:2) nucleotide in the
anneal.The18thntisthen4ntonthe3(cid:2)sideofthesubstrate, absenceofDNA-PKcsandATP(Fig.3,lanes2,4,and7).Fur-
24038 JOURNALOFBIOLOGICALCHEMISTRY VOLUME290•NUMBER40•OCTOBER2,2015
ArtemisandKuatDNAEnds
FIGURE2.ArtemisactivitycomparisononbluntdsDNAand5(cid:3)DNAoverhangs.100nM5(cid:2)32P-radiolabeledsubstratewasincubatedwith25nMDNA-PKcs
and25nMArtemisfortheindicatedtimesat37°C.Thesubstrateswerea9-ntpoly(dT)5(cid:2)overhangsubstrate(*HC85/58);a34-bp,blunt-endedsubstrate
flankedbyfourdTs(*HC57/58);anda34-ntssDNAsubstrate(*HC57).Theasterisksindicate[32P]phosphates.A,samplesfromtimepointswererunona14%
denaturingPAGE.B,cutproductswerequantified,andtheaverageoftwoindependentexperimentswasplottedasafunctionoftime.Errorbarsindicate
mean(cid:6)S.D.(Severaladditionalsimilartimecoursesgaveresultsindistinguishablefromthoseshownhere.).C,linearregressionwasusedtocalculatetheslope
andR2values.
thermore,Artemis5(cid:2)endonucleaseactivitypredominatelycut (datanotshown),althoughautophosphorylatedDNA-PKcsis
1–6ntfromthe5(cid:2)endinthepresenceofDNA-PKcs(Fig.3, requiredforallends.
lane7versuslane9),andthisactivitywasdependentonATP TitrationRevealsThatDNA-PKcsCanBeInhibitoryatHigh
(Fig.3,lane4versuslane9). Concentrations—Nextweexaminedtheeffectoftheamountof
Interestingly, Ku modulated Artemis endonuclease activity DNA-PKcsontheobservedactivityofArtemis.Wegenerateda
atbluntendsbyrestrictingthecutsto2and4ntinsteadofthe 5(cid:2)-labeled, 34-bp, blunt-ended DNA by annealing 5(cid:2)-labeled
1- to 6-nt distribution observed in Ku-independent reactions ssDNAtoitsunlabeledcomplementarystrandcontaininga5(cid:2)
(Fig.3,lane9versuslane10,5(cid:2)endresection).Theadditionof biotin (Fig. 4). This substrate was then incubated along with
Kualsostimulated3(cid:2)endonucleaseactivitybyenablingArte- streptavidin, which binds biotin tightly, to prevent/inhibit
mistocutupto9ntintothe3(cid:2)end(Fig.3,lane9versuslane10, ArtemisactionononeendofthedsDNAtoexaminetheactiv-
3(cid:2)endresection).This3(cid:2)activityremainedDNA-PKcs-depen- ity on the labeled end only. Using a blunt-ended substrate
dent (Fig. 3, lane 8 versus lane 10, 3(cid:2) end resection). Further- flankedwithfourdAs,wetitratedtheamountofDNA-PKcsin
more, the observed activities are not due to contaminating thereactionfrom2to105nM(Fig.4).KuandDNA-PKcsalone
nucleases because the catalytically inactive Artemis point did not have any detectable levels of nuclease activity (Fig. 4,
mutant,ARM14,whichwaspurifiedinthesamemanner,did lanes2and3).Artemisactivitywasmaximalata1:1molarratio
notshowany5(cid:2)or3(cid:2)activity(Fig.3,lanes10and11).Wealso withDNA-PKcs(Fig.4,lanes6–9).
confirmed that, similar to Artemis endonuclease activity on Furthermore,Artemisactivitywasstimulated(cid:4)2-to3-fold
overhangs and ssDNA (5, 6), Artemis phosphorylation is not in the presence of Ku (Fig. 4, lanes 6–9 versus lanes 10–13).
required for its endonuclease activity on blunt-ended DNA Interestingly,increasingthemolarratioofDNA-PKcstoArte-
OCTOBER2,2015•VOLUME290•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 24039
ArtemisandKuatDNAEnds
FIGURE3.ArtemisactivityonbluntdsDNAwithdTends.20nM5(cid:2)32P-radiolabeled,34-bpdsDNA(*HC57/58)wasincubatedwith50nMKu,50nMDNA-PKcs,
50nMArtemis,50nMARM14,and0.5mMATPintherespectivereactionsfor30minat37°C,followedbyresolvingthesampleona12%denaturingPAGE.
ReactionsincludedArtemis(lane2);KuandArtemis(lane3);DNA-PKcsandArtemis(lane4);Ku,DNA-PKcs,andArtemis(lane5);andKu,DNA-PKcs,andARM14
(lane6).ATPwasincludedinlanes7–11.M,markerlane;asterisk,[32P]phosphate.Thisisarepresentativegelofatleastthreeidenticalexperimentsandatleast
twopreparationsofArtemisandDNA-PKcs.(Severaladditionalsimilarexperimentsgaveresultsindistinguishablefromthoseshownhere.)
mis to 2:1 reduced Artemis activity by (cid:4)50% (Fig. 4, lane 8 exonuclease activity was observed with substrates that are
versuslane9andlane12versuslane13).Thesedatasupportthe flanked by AT but not GC base pairs at the blunt DNA ends
view that Artemis acts in a complex with DNA-PKcs at a 1:1 (Figs.3,lanes2,4,and7;4,lane4;and5, A–D,lanes1–4).The
molarratioandthatincreasingtheconcentrationofDNA-PKcs observedexonucleaseactivitywasreducedwhenKuwasadded
mayblocktheDNAendsfromArtemisactivity. (Figs. 3, lane 2 versus lane 3, lane 4 versus lane 5, and lane 7
The Blunt End DNA Sequence Determines the Extent to versuslane8;4,lane4versuslane5;and5, AandB,lanes1–4
WhichKuandDNA-PKcsStimulatethe5(cid:2)NucleaseActionof versuslanes5–8).Wehypothesizedthattheweakbasepairing
Artemis—WethenaskedwhethertheobservedKustimulation inAT-richendscomparedwithGC-richendsallowedtheDNA
is sequence-dependent at 5(cid:2) blunt ends. We generated 5(cid:2)-la- end to breathe and form transient ss/dsDNA boundaries to
beled, 34-bp, blunt-ended DNA with four dAs (Fig. 5A), four provideasubstrateforArtemis.Toexaminethesequence-de-
dTs(Fig.5B),fourdGs(Fig.5C),orfourdCs(Fig.5D)byanneal- pendentvariationfurther,wesimulatedbreathingbycreatinga
ing 5(cid:2)-labeled ssDNA with unlabeled 5(cid:2) biotinylated comple- fixed2-ntpseudo-YstructurewithdAordGunpairedssDNA
mentarystrands.Thesubstratewasthenincubatedwithpro- overhangs(Fig.6).DNAwith5(cid:2)dGswasprocessedbyArtemis
teinsasindicated,alongwithATPandstreptavidin.Artemis5(cid:2) whenforcedintoapseudo-Ystructure(Fig.6,lanes9–12versus
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ArtemisandKuatDNAEnds
FIGURE4.DNA-PKcstitrationforArtemisactivityonbluntends.20nM5(cid:2)32P-radiolabeled,34-bpdsDNA(*HC79/82)withfourdAswascreatedbyannealing
theradiolabeled(asterisk)topstrandwithitscomplementarystrandwitha5(cid:2)biotin(B).Thesubstratewasincubatedwith50nMKu,50nMArtemis,andvarious
amountsofDNA-PKcsintherespectivereactionsfor30minat37°C,followedbyresolvingthesampleona14%denaturingPAGE.Inaddition,0.5mMATPand
0.1(cid:5)Mstreptavidin(SA)wereincludedinallreactionstosuppressArtemisactivityonthe3(cid:2)end.ReactionsconsistedofKu(lane2),DNA-PKcs(lane3),Artemis
(lane4),KuandArtemis(lane5),ArtemiswithincreasingamountsofDNA-PKcs(lanes6–9),andKuandArtemiswithincreasingamountsofDNA-PKcs(lanes
10–13).Thequantified5(cid:2)resectionpercentageisnotedatthebottomofthegel.Thisisarepresentativegelofatleasttwoidenticalexperiments.(Several
additionalsimilarexperimentsgaveresultsindistinguishablefromthoseshownhere.)
lanes13–16).Interestingly,Y-structureDNAwith5(cid:2)dAswas ulatedwithGC-richbluntDNAsubstrates(Fig.5,A–D,lanes
alsostimulated2-to3-fold(Fig.6,lanes1–4versuslanes5–8). 9–12). With blunt AT ends, up to 6 nt were resected in the
These data indicate that DNA breathing may allow AT-rich presenceofDNA-PKcs(Fig.5,AandB,lanes9–12).Inaddi-
bluntDNAtobecleavedbytheArtemis5(cid:2)exonucleaseactivity tion,Kustimulatedtheendonucleaseactivity(cid:4)3-to4-fold(Fig.
intheabsenceofotherproteins(DNA-PKcsandKu). 5,AandB,lanes9–12versuslanes13–16).Conversely,forGC
Incontrast,Artemis5(cid:2)endonucleaseactivityismorecom- ends, DNA-PKcs only slightly activated 5(cid:2) exo- and endonu-
plex.Artemis5(cid:2)endonucleaseactivitywasstimulatedbyDNA- clease activities (Fig. 5, C and D, lanes 9–12). However, the
PKcswithAT-richbluntDNAsubstratesbutonlymildlystim- additionofKustimulatedArtemisactivitymorethan10-fold
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ArtemisandKuatDNAEnds
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ArtemisandKuatDNAEnds
FIGURE6.DNAendbreathingprovidesasubstrateforArtemis5(cid:3)exonucleaseactivity.ShownisthetimecourseofArtemisactivityonblunt-endedand
fixed2-ntY-structureDNA.20nM5(cid:2)32P-radiolabeled,34-bpbiotinylated-dsDNA(B)wasincubatedwith50nMArtemisand0.1(cid:5)Mstreptavidin(SA)for0,15,30,
and60minat37°C.Thesampleswereresolvedona12%denaturingPAGE.Substrateswereblunt-endeddA(*HC79/82,lanes1–4),dAY-structure(*HC98/76,
lanes5–8),blunt-endeddG(*HC80/83,lanes9–12),anddGY-structure(*HC99/84,lanes13–16).Asterisk,[32P]-phosphate.Thequantified5(cid:2)resectionpercent-
ageisnotedatthebottomofthegel.Thisisarepresentativegelofatleasttwoidenticalexperiments.
(Fig.5,CandD,lanes9–12versuslanes13–16).Insummary, KustimulatedArtemisactivityonly3-to4-foldanddiminished
KucouldmodulatetheactivityofArtemis(cid:2)DNA-PKcsdepend- thediversityofresection.Specifically,1-to6-ntresectionwith-
ingontheAT-orGC-richnessofbluntends.ForAT-richends, outKuand1to2ntwithKu(Fig.5,AandB,lanes9–12versus
FIGURE5.TheTimecourseof5(cid:3)endonucleaseactivityonblunt-endedDNArevealsthatArtemisresectsupto6nt,requiresDNA-PKcs,andis
modulatedbyKutopreferentiallycut2ntintotheduplex.20nM5(cid:2)32P-radiolabeled,34-bpbiotinylateddsDNA(B)wasincubatedwith50nMKu,50nM
DNA-PKcs,and50nMArtemisfor0,15,30,and60minat37°Cinthelanesasindicated.Inaddition,0.5mMATPand0.1(cid:5)Mstreptavidin(SA)wereincludedin
allreactionstosuppressArtemisactivityonthe3(cid:2)end.Thesampleswereresolvedona14%denaturingPAGE.M,markerlane;asterisk,[32P]phosphate.The
quantified5(cid:2)resectionpercentageisnotedatthebottomofthegel.Thisisarepresentativegelofatleastthreeidenticalexperimentsandatleasttwo
preparationsofArtemisandDNA-PKcs.(Severaladditionalsimilartimecoursesgaveresultsindistinguishablefromthoseshownhere.).A,5(cid:2)dAsubstrate
(*HC79/82).B,5(cid:2)dTsubstrate(*HC57/76).C,5(cid:2)dGsubstrate(*HC80/83).D,5(cid:2)dCsubstrate(*HC81/84).
OCTOBER2,2015•VOLUME290•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 24043
ArtemisandKuatDNAEnds
lanes13–16).Incontrast,KustimulatedArtemisactivitymore breathingrequirement.Thisallowsthe3(cid:2)overhangresection
than 10-fold in GC-rich ends overall, with the most frequent tobesequence-independent.
resectioneventsat1–2nt(Fig.5,CandD). ArtemisResectionProgressesInternallyalongtheDNAwith
Artemis3(cid:2)EndonucleaseResectionDoesNotShowSubstan- Time—BecauseweestablishedthatArtemisfirstactsonthe5(cid:2)
tialSequenceDependencebutIsStimulatedbyDNA-PKcsand endpriortoactingontheresulting3(cid:2)overhang,wewondered
Ku—Wethenaskedwhetherthesequence-dependentactivity whether this process would repeat until the DNA was pro-
isalsoobservedonthe3(cid:2)endofblunt-endedDNA.3(cid:2)-labeled, cessed completely. We initially showed that a 34-bp, blunt-
34-bp, blunt-ended DNA flanked by four dAs (Fig. 7A), four endedDNAcouldbeprocessedupto11ntafter2h(Fig.8A,
dTs (Fig. 7B), four dGs (Fig. 7C), or four dCs (Fig. 7D) was lane5),whichwouldleave(cid:4)23bpremaining.Wethenincu-
generatedbyfill-insynthesiswiththeKlenowfragmentofDNA bateda73-bpDNAwithKu,DNA-PKcs,andArtemisforupto
polymeraseI(3(cid:2)35(cid:2)exo(cid:3))and[(cid:4)-32P]dNTP.Thesubstrate 160 min (Fig. 8B, lanes 1–6). The resulting gel showed that
wasthenincubatedwithKu,DNA-PKcs,andArtemis,asindi- Artemis endonucleolytically resected up to 4 nt on the 5(cid:2)
cated,inthepresenceofATPandstreptavidin.Theunlabeled end(Fig.8B,lane6,5(cid:2)endresection).Processingonthe3(cid:2)end
complementary strand contained an unlabeled 5(cid:2) phosphate proceeded until (cid:4)25–30 nt remained (Fig. 8B, lane 6, 3(cid:2) end
anda3(cid:2)biotintoblockArtemisactiononthe3(cid:2)end.3(cid:2)exonu- resection).Becauseweknowthat(cid:4)26bparerequiredformax-
cleaseactivitywasobservedonlyonthe3(cid:2)dADNAandslightly imal Ku-dependent DNA-PKcs activity (17), it is likely that
on3(cid:2)dT,dG,anddCDNA(Fig.7,A–D,lane2).Interestingly, ArtemisresectionproceedsuntiltheDNAislongenoughfor
Kudidnotblockthis3(cid:2)exonucleaseactivity(Fig.7,A–D,lane2 oneKu(cid:2)DNA-PKcscomplextoremainboundtotheDNA.
versuslane4).Artemisactivityonthe3(cid:2)endwasonlymildly
Discussion
stimulatedbyDNA-PKcsandproducedadistributionofcuts
Underphysiologicalconditions(ArtemisplusDNA-PKcsin
upto5ntintotheduplexportionofthesubstrate(Fig.7,A–D,
Mg2(cid:5) solutions), we found that resection at blunt DNA ends
lane2versuslane8).KufurtherstimulatedArtemisactivity6-
dependsontheDNAsequence.However,whenKuisalsopres-
to8-foldindependentofsequence(Fig.7,A–D,lane8versus
ent,thedisparityiseliminated,andbluntendsofallsequences
lane12).
areresectedefficiently.Thisisthefirstbiochemicaldocumen-
Interestingly,theseresultsareincontrasttoArtemisactivity
tation showing that Ku can affect the nucleolytic behavior of
observedonthe5(cid:2)end.Forone,thebreathingrequirementwas
Artemis. Further study of it has revealed major unifying fea-
notsequence-dependentonthe3(cid:2)end.Themildactivitystim-
turesoftheArtemisnuclease.
ulationbyDNA-PKcsonthe3(cid:2)end,inwhichonlyupto8%of
UnifyingKineticFeaturesforArtemisActionatDNAEnds—
the substrate was cut after 60 min (Fig. 7, A–D, lane 8), was
PreviousworkonArtemishasdocumenteditsactionatss/ds-
similartothestimulationobservedonGC-rich5(cid:2)ends(Figs.5,
DNA boundaries, specifically at hairpins and overhangs and
CandD,lane12,and7,A–D,lane12).Secondly,theadditionof
gapsandflaps(7,11).Onestudyalsodescribedendonuclease
KustimulatedArtemisactivity6-to8-foldonthe3(cid:2)endinde- actionbyArtemisaloneinMg2(cid:5)solutionsatbluntDNAends
pendent of sequence (Fig. 7, A–D, lane 8 versus lane 12),
(18).Butthislatterfindingwascuriousbecausenootherlabo-
whereasArtemisactivityon5(cid:2)endswassequence-dependent(a
ratoryhasfoundendonucleaseactivityofArtemisaloneunder
3- to 4-fold increase for AT-rich and a more than 10-fold Mg2(cid:5) conditions. We noted that numerous freeze and thaw
increaseforGC-rich).
cyclesofstoredArtemisenzymepreparationseventuallycause
5(cid:2) Resection Occurs Prior to 3(cid:2) Resection on Blunt DNA
such behavior, suggesting a denaturation-induced change in
Ends—We determined that Artemis performs asymmetric
theenzymethatpartiallyeliminatestherequirementforDNA-
resectionforthe5(cid:2)versus3(cid:2)strands.Resectiononthe5(cid:2)strand PKcs. In addition, Artemis alone can cut overhangs in Mn2(cid:5)
at AT-rich blunt ends occurred in a largely Ku-independent
solutions, suggesting a permanent conformational change of
manner.GC-rich5(cid:2)endsweremildlystimulatedbyDNA-PKcs
theenzymeinthepresenceofthisdivalentcation(6,9,19).For
andstimulatedstronglybyKu.Conversely,Artemisexhibited
thisreason,evaluationofArtemisunderconditionswhereitis
sequence-independentactivityon3(cid:2)endsthatrequiredKufor dependent on DNA-PKcs was necessary to consider its func-
robustactivity.Becauseitappearedthat5(cid:2)endresectionwas tion under physiologic conditions at blunt DNA ends and to
more efficient, we asked whether 5(cid:2) end processing was integratethisintothespectrumofArtemisnucleaseactivities.
requiredpriorto3(cid:2)endprocessing.Weannealeda5(cid:2)-labeled, OurfindingofresectionbyArtemisaloneinMg2(cid:5)solutions
34-ntoligotoits3(cid:2)biotinylatedcomplementaryoligowithor atAT-richDNAendsbutnotatGC-richDNAendssuggested
without10phosphorothioatebondsonthe5(cid:2)end.Aphospho- thatDNAendbreathingisrequiredforArtemisaction.Inother
rothioate bond, formed by the sulfurization of a nonbridging studies, we have documented the increased breathing of AT-
oxygen in the phosphodiester bond, suppresses the action of rich DNA ends compared with GC-rich ends (20). When we
nucleases.WeperformedatimecourseofArtemisactivityin “forced”breathingherebycreatingafixed,2-ntY-structurein
thepresenceofDNA-PKcsandKu.WeobservedthatArtemis which the 2 nt arms of the Y cannot anneal, we found that,
activityonthe3(cid:2)endofdsDNAsubstratewasreducedsignifi- indeed, the DNA sequence of the arms no longer matters
cantly when the complementary strand was unable to be cut becausetheendsareinapermanentssDNAconfiguration.
first(Fig.8A,lanes1–5versuslanes6–10).Therefore,itappears ArequirementforendbreathingforArtemistoactonablunt
that the 5(cid:2) strand resection occurs first, always leaving a 3(cid:2) endexplainswhysuchendresectionbyArtemisisslowerthan
ssDNAoverhangthat,byitsssDNAnature,doesnothaveany overhangresection.Thebreathingstepisrequired,andtheend
24044 JOURNALOFBIOLOGICALCHEMISTRY VOLUME290•NUMBER40•OCTOBER2,2015
ArtemisandKuatDNAEnds
FIGURE7.TheTimecourseof3(cid:3)endonucleaseactivityonblunt-endedDNArevealsthatArtemisresectspredominantly1–3nt,requiresDNA-PKcs,
andisstimulatedbyKu.20nM3(cid:2)32P-radiolabeled,34-bpbiotinylateddsDNA(B)wasincubatedwith50nMKu,50nMDNA-PKcs,and50nMArtemisfor0,15,
30,and60minat37°Cinthelanesasindicated.Inaddition,0.5mMATPand0.1(cid:5)Mstreptavidin(SA)wereincludedinallreactionstosuppressArtemisactivity
onthe5(cid:2)end.ThePonthebottomstranddenotesacoldphosphategrouponthe5(cid:2)end.Thesampleswereresolvedona14%denaturingPAGE.M,marker
lane;asterisk,[32P]phosphate.Thequantified3(cid:2)resectionpercentageisnotedatthebottomofthegel.Thisisarepresentativegelofatleastthreeidentical
experimentsandatleasttwopreparationsofArtemisandDNA-PKcs.(Severaladditionalsimilartimecoursesgaveresultsindistinguishablefromthoseshown
here.).A,3(cid:2)dAsubstrate(HC86*/89).B,3(cid:2)dTsubstrate(HC74*/73).C,3(cid:2)dGsubstrate(HC87*/90).D,3(cid:2)dCsubstrate(HC88*/91).
OCTOBER2,2015•VOLUME290•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 24045