eigUiest,Biig107,China. 100871, Beijing University, Peking igigLi repair Tingting damage DNA regulates of and ubiquitylation H2A antagonizes neddylation H2A RNF168-mediated ARTICLE RESEARCH 2238 2014 February 13 Accepted 2013; July 22 Received ` al., et Tateishi 2002; al., Liakopoulos et Ou 1998; 2000; in 1 al., al., et 1998; et processes Osaka Lammer (Hochstrasser, 1998; developmental 2000; al., mouse et Candido, to and and yeast viability Jones from of ranging al., roles cell et essential was organisms the in (Kerscher demonstrated that pathway have NEDD8 conjugation studies gene to (UBL) Genetic distinct identity protein 2006). sequence a like a acid ubiquitin but amino (Kamitani the greatest ubiquitin as of the development member has NEDD8 a discovered family, brain As 1997). mouse first al., et during was downregulated (NEDD8) downregulated developmentally 8 expressed cell precursor Neural INTRODUCTION H2A, RNF168 repair, Ubiquitylation, damage DNA the Neddylation, WORDS: through KEY repair damage DNA the a pathway. to suggest elucidate neddylation approach and findings neddylation, modulatory and Our novel ubiquitylation H2A BRCA1. of protein relationship response ubiquitylation DNA the damage and of the suppression H2A regulates through of process basal negatively repair to NEDD8 damage decreased Mechanistically, neddylation ubiquitylation H2A levels. repair, whereas damage an gradually, of with stages increased later damage decreased DNA the facilitates neddylation During which H2A, repair. H2A of ubiquitylation of the to in level RNF168 increase response E2 the in its of Moreover, damage, UBE2N). and Inhibition as DNA RNF168 known activity. (also between Ubc13 ubiquitin interaction enzyme E3 the impairs its neddylation for RNF168 H2A of necessary neddylation Interestingly, and promotes NEDD8, is neddylation. for substrate NEDD8 and RNF168 a itself ligase ubiquitylation is free RNF168 E3 H2A of the both that level promotes H2A found we decreased of covalently Furthermore, neddylation a ubiquitylation. is that and of NEDD8 ubiquitylation and suppresses H2A, that (H2A), NEDD8 ubiquitylation. 2A show its antagonizes histone the we remain to biological and pathways Here, conjugated many NEDD8 neddylation, unknown. and in for ubiquitin largely factor the substrates between regulatory the relationship important However, an processes. is NEDD8 ABSTRACT Teeatoscnrbtdeulyt hswork this to equally 100871, contributed Beijing authors University, *These Peking Sciences, Life China. of School Biology, Molecular uhrfrcrepnec ([email protected]) correspondence for Author tt e a fPoenadPatGn eerh colo ieSciences, Life of School Research, Gene Plant and Protein of Lab Key State 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,23–28doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. c 1,2, 2X hc ute lcstercuteto the of recruitment the blocks further which H2AX, ,JnogGuan Junhong *, 2 eateto iceityand Biochemistry of Department 1,2, ,Zj Huang Ziji *, 2 in Hu Xiang , 2 ta. 04.Ndyaini lorpre opoetribosomal protect to Xirodimas reported (Dohmesen 2007; also al., is pathway et Neddylation Singh the 2011; 2004). of al., al., et et stages Noguchi and 2008; several al., Wimuttisuk NF- et at increased substrates 2004; and neddylation in suppresses al., activity, by of p53 et transcriptional of results degradation neddylation Pan its MDM2-mediated and 2001; 2007). the al., Singer, enzyme and et recruitment conjugating (Kawakami promotes activity E2 complex, ubiquitylation ligase an Skp–Cullin–F- the ubiquitin E3 of of (SCF) proteins ubiquitin scaffold box regulating stability. cullins, protein a of in and activity Neddylation indicate factor NEDD8 transcription mechanisms activity, of ligase molecular function into widespread Investigations 2001). ojgtn nyeUC2 loihbt 2 n H2AX and H2A inhibits NEDD8- also by the of UBC12, neddylation ubiquitylation. mutants RNF168 enzyme target or a of NEDP1, conjugating at enzyme also downregulation BRCA1 is deneddylating of and the RNF168 recruitment Moreover, NEDD8, the sites. of blocks repair and damage ubiquitylation H2AX, competes DNA suppresses and which NEDD8 H2A RNF168, that H2A. of report by of we mediated Here, ubiquitylation is 2013). with al., tail H2A et N-terminal of (Ma the RNF111 at neddylation repair 2012; ligase neddylated al., E3 strongly et the is damage are by H4 (Guzzo 2012; 2013). NEDD8 response al., DNA damage and et al., DNA SUMO Ma the of BRCA1. et in and involved 2008), recruitment RAP80 Mattiroli also al., as et 2007; the Plans such 2009; proteins, al., al., facilitates et Huen et Pinato 2012; which 2012; al., Mailand al., et et Gatti RNF2), 2007; Oestergaard 2004; as al., al., known via et variant et (also (Fang chains its RNF168 RING2 polyubiquitin and including and RNF8 K63-linked ligases, (H2A), E3 by 2A multiple modified Histone are al., pathway. H2AX, et repair (Zuo damage degradation and ubiquitylation 2013). blocks TGF- K48-linked of the and its promotes conjugation c-Cbl of al., ubiquitylation degradation. modifications et neddylation mediates ubiquitin-directed both (Xirodimas antagonizes from p53 and that substrates of Neddylation p53, example, activity ligase to 2004). transcriptional For NEDD8 and both the E3 two manner. and suppress ligases an for these similar ubiquitin E3 both a that is specificity Some in suggesting MDM2 dual function time. neddylation, modifications same have modifications and both the two ubiquitylation enzymes be these at can deconjugating and substrates occur neddylated, Most usually question. and intricate ubiquitylated an Zhang remains 2008; al., et 2012). (Xirodimas al., et destabilization from proteins n ioegZheng Xiaofeng and bqiyaini neryadipratsga nteDNA the in signal important and early an is Ubiquitylation pathways NEDD8 and ubiquitin the between relationship The 1,2, b ` yeI eetradsuppresses and receptor II type k ciiyi controlled is activity B

Journal of Cell Science rnfce ihHsuiutno i–ED,wt rwtot3lgND1 hnteedgnu edlto n bqiyaino 2,H n 4were H4 and H3 H2B, of ubiquitylation and neddylation endogenous blotting. the western then and 3Flag–NEDP1, down without pull or His-tag with by His–NEDD8, detected or His–ubiquitin with transfected itnsapae ihahg bnac n frequency. and abundance that high found that and a substrates laboratories with different neddylation appeared by for potential identified histones al., substrates et been analyzed Xirodimas 2008; new had 2006; al., We Shiekhattar, and identify et Norman 2008). (Jones 2006; to al., approaches et tried Li proteomics using have neddylation neddylation groups for substrates are H4 Several and H2B H2A, histones The RESULTS ARTICLE RESEARCH edlto.Teefc fMN94o h edlto n bqiyaino xgnu 2 tppnl,adedgnu 2 n / 3 and without H2A The endogenous or and and panel). with H2A panel), panel) treated downregulates (top (lower been ubiquitylated H2A MLN4924 co-immunoprecipitation of exogenous had with or of amount that Treatment ubiquitylation panel) cells and (B) top HEK293T neddylation lysate. down, in the pull whole-cell examined on (His MLN4924 WCL, down of immunoprecipitation; effect pull IP, The His-tag blotting. neddylation. through western enriched by neddylated. were detected are proteins then H4 modified and and HA–NEDD8, H2B or H2A, NEDD8 histones The 1. Fig. and H2A endogenous detected we MLN4924 confirm H2A, inhibited. further that of To be left). neddylation revealed 1B, could (Fig. the analysis neddylation H2A to H2A pulldown inhibitor, suppressed of E1 His-tag but neddylation neddylation Indeed, UBA1) specific We whether as 2012). a al., examine known MLN4924, et (Hjerpe used (also on enzyme next Ube1 E1 relies NEDD8-activating enzyme NEDD8 the is not E1 It overexpressed 1A). ubiquitin of (Fig. the conjugation neddylated clearly that was reported H2A be that pulldown can His-tag showed histones and assays whether co-immunoprecipitation material histones, investigated Both (supplementary therefore, neddylated. to nucleus We, the similar S1A). in Fig. that, existed mainly indicated NEDD8 analysis Immunostaining x xsnmescreaewt h ubrdlnsi h idepnl C noeosHBadH r edltd E23 el were cells HEK293T neddylated. are H4 and H2B Endogenous (C) panel. middle the in lanes numbered the with correlate numbers axis c 2Xwsqatfe yuigteOysyIfae mgn ytm(ICRBocecs ntreidpneteprmns(bottom experiments independent three on Biosciences) (LI-CDR System Imaging Infrared Odyssey the using by quantified was H2AX A 2 sndyae.HK9Tclswr rnfce ihFa–2,Hsuiutn(b,His– (Ub), His–ubiquitin Flag–H2A, with transfected were cells HEK293T neddylated. is H2A (A) m L42 o 4h n hnsbetdt i-a uldw n etr ltig The blotting. western and down pull His-tag to subjected then and h, 24 for MLN4924 M ojgto fuiutnt 2 and H2A to ubiquitin of conjugation variant its eetda oiiecnrl efudta both that found of ubiquitylation NEDD8 We of Interestingly, conjugation right). control. the 1B, blocked (Fig. MLN4924 positive and with treatment a H2A endogenous as detected aae n oas nesadtecreainwt H2A with DNA correlation of the VP16-induced mechanism understand in thus the also function we elucidate to ubiquitylation. its to response, and and H2A been damage damage, on has neddylation DNA function work damage H2A in the important our the DNA Recently, modification to focused to owing chromatin. H2A and and response 2013), the of H3 al., in on et H2A, (Ma and H4 NEDD8 reported that of of ubiquitylated indicate targets neddylation data both histone are These the were H4 NEDP1 from 1C). NEDD8 deneddylase (Fig. H4 deconjugated the substrates SENP8) and and as not, known was H2B (also H3 whereas H4. neddylated, and These H3 1B). neddylated. (Fig. is MLN4924 H2A with that indicate treatment data after reduced also et eeaie h noeosmdfcto fH2B, of modification endogenous the examined we Next, ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal c 2Xb i–ED ulonasy the assay, pulldown His–NEDD8 by H2AX c 2Xwr edltdadthat and neddylated were H2AX c 2X(idepnl,was panel), (middle H2AX c 2Xwsalso was H2AX c H2AX c 2Xwas H2AX 2239 and

Journal of Cell Science ekatvt,weesRF upesdteconjugation the suppressed RNF8 strongly whereas a RNF168 exhibited activity, that RING2 H2A. showed to weak NEDD8 results to of including reported conjugation The are the – promoted ligases activity. ligases E3 many dual E3 as have – ubiquitin RING2 and we H2A RNF168 H2A, of RNF8, reported neddylation the the for required examined a ligase E3 is the NEDP1 identify and To H2A, for ligase E3 deneddylase neddylation a is RNF168 ARTICLE RESEARCH niae lsis n hnteefcso idtp W)ND1adtectltclyiatv 13 EP uato 2 edlto eedtce b detected wit were neddylation transfected 2240 H2A were on cells mutant HEK293T NEDP1 activity. C163S inactive deneddylating blotting. catalytically investigate its the were western attenuates and ubiquitylation wer and NEDP1 H2A activity cells (WT) down or HEK293T NEDP1 wild-type neddylation neddylation. pull of of H2A H2A tag effects on Downregulation of the USP7 deneddylase (D) then or a and NEDP1 blotting. is plasmids, of RNF168 western indicated NEDP1 expression against and (C) the 2) blotting. of and down effects western 1 pull the and (shRNA tag and RNAs down plasmids, hairpin pull indicated small His-tag t the different performed by with and were detected transfected His–NEDD8 analyses was with blot (H2A-N8) transfected western neddylation were and H2A cells down HEK293T then pull H2A. His-NEDD8- His-tag endogenous mutant then of NEDD8 h, neddylation the 48 reduces deneddylase. with for the Transfection Flag–RING2 is neddylation. or NEDP1 H2A HA–RNF168 and Flag–RNF8, detect H2A, His–NEDD8, for with ligase transfected E3 were neddylation cells the is RNF168 2. Fig. to ligase E3 an as H2A. functions of RNF168 neddylation the that As 2B). mediate (Fig. suggest down. H2A data of knocked neddylation These endogenous (RNAi) variation was the interference RNA weakened the RNF168 using clearly of by assessed RNF168 role when of the next ablation neddylation expected, confirm we further H2A neddylation, To in H2A control. lysines in negative to a conjugated RNF168 as be used cannot subsequently, was and, NEDD8- acids amino A 2A). (Fig. D Gmtn htlcstels two last the lacks that mutant GG D Gsre sangtv oto.WL hl-ellst.()Dpeino RNF168 of Depletion (B) lysate. whole-cell WCL, control. negative a as served GG osrce eiso uat nwihLs1 n/rLys120, and/or Lys119 which we in conjugated, mutants is of NEDD8 which series to a H2A constructed on ubiquitin sites and the NEDD8 examine by To competitively modified is H2A htUP ih lofnto sadndyaefrH2A. for deneddylase and a H2A as for function deneddylase also 2C). a might (Fig. is USP7 NEDP1 whereasthat conjugations that ubiquitin conjugation, indicate results affected NEDD8 These weakly weakened only USP7 NEDP1 exogenous Interestingly, neddylation. of examined H2A also on expression we USP7 2010), al., of H2A et effect Maertens of the 2010; of ubiquitylation al., suppress et Bie to level specific (de reported H2A been ubiquitin the the has Because (USP7) NEDP1, 2D). 7 and in protease (Fig. increased impaired serine was to was neddylation mutated catalytic activity was the 163 When deneddylation position 2C). (Fig. at H2A cysteine from NEDP1 NEDD8 Indeed, H2A. deconjugated for deneddylase a is NEDP1 whether address EP saseii eedltn nye etu re to tried thus we enzyme, deneddylating specific a is NEDP1 A N18ctlzstendyaino noeosHA HEK293T H2A. endogenous of neddylation the catalyzes RNF168 (A) ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal the h yHis- by d His- y e o ,

Journal of Cell Science 19/10 uatcudntcmltl bls H2A the abolish mutants that completely single probable not is K120R could It and S2B). mutant K119R Fig. K119R/K120R the mutant material neddylation double ubiquitylation both H2A (supplementary K119R/K120R whereas in H2A S2A), the increased that Fig. by to material showed abolished (supplementary mutated were mostly ubiquitylation results were and was neddylation The sites, of levels investigated. conjugation the and ubiquitin arginine, reported two ARTICLE RESEARCH el eetasetdwt aiu lsis sidctd n hntetdwt rwtot3 was without HEK293T H2A or ubiquitylation. of with H2A ubiquitylation treated on and NEDP1 then neddylation and and UBC12-C111A UBC12-C111S, the indicated, MLN4924, bl and as of western plasmids, plasmids, effects by indicated various The analyzed the (C) with were with blotting. transfected (WCL) western transfected were lysates and were cells whole-cell down cells and pull HEK293T His-tag His–NEDD8, (B) by or H2A. examined His–ubiquitin against Flag–H2A, antiboday with an transfected using were cells HEK293T ubiquitylation. (Ub). antagonizes ubiquitin neddylation Histone 3. Fig. was the H2A with modified ubiquitylated been has endogenously that H2A above represents H2A, which with band detected, co-expressed was extra NEDD8 Dayhoff, an and or Hunt cells, ubiquitin in 1975; When al., of resting is 1977). 10–15% et (Goldknopf that which in noted ubiquitylated is have ubiquitin, 3A, H2A that Fig. endogenous reports previous by with in accordance ubiquitylated shown was As H2A ubiquitylation. relationship H2A antagonistic an at neddylation. that neddylated and ubiquitylation or suggests is between K119 exists when mutated H2A H2A was that of neddylation K120 indicate increased The results material sites. multiple These (supplementary not S2B). could neddylation truncated, C-terminal mutation Fig. H2A was the tail The at abolish C-terminal lysines 2012). the completely five or the al., the mutated at all were et tail either ubiquitin (Mattiroli which of residues in constructs, conjugation K15 and the K13 of because ubiquitylation eepromd(o ae) h muto bqiyae 2 a uniida ecie nFg B(otmpnl.Teqatfcto aasonare shown data quantification The panel). (bottom 1B Fig. in described the as on quantified shown was numbers The H2A mean experiments. ubiquitylated independent of three amount of The representative panel). (top performed were enx netgtdwehrND8de,ide,influence indeed, does, NEDD8 whether investigated next We 6 ... * s.e.m., P , .5 ** 0.05, P , .1 *** 0.01, P , 0.001, n 5 3. A noeos(no 2 bqiyaini niie yetpcepeso fND8(8 and (N8) NEDD8 of expression ectopic by inhibited is ubiquitylation H2A (Endo) Endogenous (A) x xscrepn otenme ae ntetppnl ausaesona the as shown are Values panel. top the in lanes number the to correspond axis h ED ojgto aha wn oteeitneof existence abolish, the to completely owing whereas not pathway ubiquitin, could conjugation to NEDD8 but mutants NEDD8 two the These free suppressed, 2000). al., of et dominantly E2 (Wada ratio NEDD8 not neddylation does to the UBC12-C111A lack bind to reduce can known UBC12-C111S and however, are activity; mutants enzyme Both C111S mutants, C111A. neddylation UBE2M) as and known (also H2A UBC12 two constructed suppressed also ectopic ubiquitin the 3B). (Fig. consistently, of NEDD8 and, of molecules expression ubiquitylation, overexpression small expected, H2A two As these suppressed modification. of and effects H2A ubiquitin co- ectopic the on HA-tagged we analyzed or the finding, and His- this from NEDD8 with confirm resulting together further H2A ubiquitin, To transfected altered to NEDD8. that the H2A of NEDD8 of cells expression free because is in promoted of endogenous This ubiquitylation ratio 2A). RNF168 (Fig. H2A H2A that NEDD8 both suppressed overexpressed for neddylation, observation and ligase overexpressed E3 neddylation and that an the cells as Consistently, ubiquitylation in to 2A). inhibited (Fig. NEDD8 was similar ubiquitylation H2A was 3A). (Fig. ubiquitylation This H2A endogenous ubiquitin suppressed expressed NEDD8 exogenously or and NEDD8, or ubiquitin tagged noeosUC2poen.A hw nFg C h inhibition the 3C, Fig. in shown As proteins. UBC12 endogenous oepoeti idn ne hsooia odtos we conditions, physiological under finding this explore To ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal m L42 o 4h i-a uldw n etr blotting western and down pull His-tag h, 24 for MLN4924 M 2241 otting

Journal of Cell Science EERHARTICLE RESEARCH 2242 4. Fig. e etpg o legend. for page next See ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal

Journal of Cell Science bqiyaini nrae,wihi eivdt aiiaethe H2A facilitate of to level believed the the is contrast, during which By removed increased, damage. is is and DNA ubiquitylation cells of resting stages and in early H2A ubiquitylation to was conjugated the 4B). that (Fig. result data on a previous obtained our VP16 and with the H2A consistent with examined endogenous further of treatment neddylation We overexpression of increase. the this whereas effect suppressed VP16, NEDD8 with of treatment to response i-bqii rHsND8wr rae iho ihu 50 without or with and treated Flag–H2A were with His-NEDD8 transfected or been His-ubiquitin had that cells HEK293T response. neddylation. and damage H2A DNA induces the damage regulates Neddylation 4. Fig. ARTICLE RESEARCH nrae 2 bqiyainbtdcesdHAneddylation H2A to decreased led but VP16 4A). (Fig. with ubiquitylation treatment H2A that increased showed results The conditions. and H2A of repair Ubiquitylation damage in DNA modulates resulted Neddylation ubiquitin. the thus, and NEDD8 demonstrate and, between data reduced relationship These NEDD8 competitive 3C). UBC12-C111S the (Fig. free a ubiquitylation H2A of endogenous showed increased of Expression UBC12-C111S amount whereas effect. effect, weaker a the H2A inhibitory had suppressed or UBC12-C111A mutants, – MLN4924 greater UBC12 mutants two with UBC12 the treatment Of the ubiquitylation. or by NEDP1 – of pathway expression NEDD8 the of Fa–B1-11,adtesbellrlclzto fteDNA-damage- the proteins of related localization subcellular Flag- or the the 3Flag–UBC12-C111S and or mutants 3Flag–UBC12-C111A, HA–NEDD8 UBC12 with inactive transfected catalytically were tagged cells and HeLa NEDD8 of sites. (E) accumulation (ctrl). damage the control a inhibit as mutants served UBC12 vector empty the with panel) Transfection (top H2A endogenous of c ubiquitylation 50 min, without 60 or or and with min treated 30 were been for plasmids had VP16 indicated that the cells HEK293T with ubiquitylation. transfected suppress H2AX UBC12 down deficient and pull catalytically H2A His-tag and VP16-induced NEDD8 and by (D) neddylation points collected blotting. H2A time western were different addition. and cells at drug the after examined and were h, out 24 ubiquitylation washed or then 12 was or 50 drug immediately, to were exposed the and cells h, h HEK293T 1 48 damage. for for DNA His–Ub to or ubiquitylation His–NEDD8 response with and in transfected panel) H2A western (top of by neddylation panel) followed the (bottom down in pull Changes His-tag (C) by blotting. or detected with were treated H2A then endogenous h, 24 for were 50 His–NEDD8 cells without HEK293T or VP16 H2A. His–ubiquitin with endogenous with treatment of transfected of neddylation effect and Endo, The ubiquitylation blotting. (B) on then western lysate. were by whole-cell H2A followed WCL, of down endogenous; 3 neddylation pull with and His-tag treated Ubiquitylation by been control. investigated had a that as cells used min, were 60 or min 30 n igesrn ras n xmndteuiutlto of ubiquitylation the examined and double- and DNA H2A breaks, induce can NEDD8- single-strand that or drug ubiquitin- and a the VP16, treated with We cells neddylation process. transfected this whether in in role interested a were plays We repair. damage DNA ihteepyvco evda oto.Etpc tiigo h ectopically the of 10 staining bars: Ectopic, Scale control. a proteins. as expressed served vector empty the with against antibodies with c staining after microscopy immunofluorescence mgscpue yuigteLM70NOadDocnmicroscope DuoScan and NLO right). 710 the on LSM on based the (panels quantification using for by used captured was images software analysis image Volocity 2Xuiutlto a uniida ecie nFg B(ih panels). (right 1B Fig. in described as quantified was ubiquitylation H2AX 2XadBC1 h uliaeidctdb AIsann.Transfection staining. DAPI by indicated are nuclei The BRCA1. and H2AX h frmnindfnig niaeta ED is NEDD8 that indicate findings aforementioned The c 2X(otmpnl eedtce ywsenbotn.Teaon of amount The blotting. western by detected were panel) (bottom H2AX m c P6fr3 i r6 i,uiutlto n edlto of neddylation and ubiquitylation min, 60 or min 30 for VP16 M 2X n h edlto fHAudrdifferent under H2A of neddylation the and H2AX, c 2Xuiutlto a ral nrae in increased greatly was ubiquitylation H2AX c 2XadBC1wr nlzdb using by analyzed were BRCA1 and H2AX c 2Xuiutlto u erae H2A decreases but ubiquitylation H2AX c m 2Xi niprateetin event important an is H2AX m( c 2X;5 H2AX); c 2XadBC1a DNA at BRCA1 and H2AX m BC1.The (BRCA1). m m L42 o 4h 24 for MLN4924 M m P6for VP16 M A DNA (A) m VP16 M m M entetdwt P6 sexpected, As VP16. with treated been on C111A and C111S ectopically mutants of UBC12 VP16-induced damage effect the the or DNA NEDD8 investigated expressed we downstream Next, factors. recruits response negatively which is process. repair polyubiquitylated, process the repair terminate to damage neddylation H2A H2A to DNA by reasonable of is regulated the it Therefore, ubiquitylation that 4C). (Fig. whereas conclude level basal gradually, the neddylation of to increased the returned stages VP16, later H2A with At treatment of by proteins. induced repair damage, damage DNA DNA of recruitment 1,SC.W lotse h feto edlto nthe on neddylation of in effect Figs the obtained material tested of (supplementary were also between localization cells subcellular We results relationship HeLa S2C). Similar and S1C, competitive cells NEDD8. the HCT116 and with UBC12-C111A ubiquitin which panel). correlates UBC12-C111S, than lower of effect also 4D, inhibitory level stronger (Fig. the a with exhibited analysis consistent blot was western This ubiquitylated 3C. conjugation, Fig. endogenous ubiquitin in on described NEDD8 of as effect suppressive the from inhibited NEDD8 of overexpression c that showed assay pulldown aeydtce ncnrlcls(upeetr aeilFig. material (supplementary cells of accumulation control The S1B). in detected barely ciiyi epnet N aae N18strongly RNF168 and RNF168-mediated damage. H2A inhibited of NEDP1 ligase whereas DNA ubiquitylation VP16, E3 with to cells of ubiquitin and response H2A RNF168 promoted 5B). the in that (Fig. activity assessed to found activity ligase NEDD8 we E3 and co-transfected ubiquitin of Additionally, its NEDP1 conjugation We impaired and the and H2A. RNF168 abolished RNF168 of substantially with NEDP1 ubiquitylation al., cells et the Pan HEK293T of neddylation 2000; on the al., of influence et RNF168 the Osaka examined we both 2001; therefore, al., 2004); were et 5A). (Kawakami ligases (Fig. activity there protein E3 RNF168 proteins, modified these these the of of amount of greater that, a all and was neddylated. neddylated, that are and – showed ubiquitylated RNF168 and results RNF8 The RING2, including of ligases – E3 the H2A are whether To examined that ligases. we E3 proteins hypothesis, H2A this other as possibly, the investigate such of response, damage neddylation is, DNA the the or in This of involved inhibition NEDD8. the MLN4924, to and due ubiquitin with between cells relationship pathway H2A/ prevents mutants) neddylation of UBC12 c the the or treatment NEDP1 of of overexpression the inhibition that (through activity ligase observation E3 its The regulates and RNF168 ubiquitylated of and neddylation neddylated both is RNF168 B1 uat 11 n 11 Fg E,wihis H2A which of experiments ubiquitylation 4E), Our the (Fig. 3C. antagonizes Fig. and C111A in neddylation shown that and results show the the C111S or with NEDD8 consistent overexpressed mutants that cells UBC12 in decreased sites repair N18E iaeatvt Fg D.Tkntgte,teedata these together, Taken 5D). (Fig. suppressed activity also ligase E3 mutants RNF168 UBC12-C111S and UBC12-C111A the ugsigta edlto euae N aaerepair. damage DNA regulates strongly neddylation histones, that these of suggesting ubiquitylation decreases also mutants, 2Xuiutlto em ocnrdc h competitive the contradict to seems ubiquitylation H2AX 2Xuiutlto Fg D pe ae) hc resulted which panel), upper 4D, (Fig. ubiquitylation H2AX nrsos oDAdmg,HAand H2A damage, DNA to response In thsbe eotdta edlto euae 3ligase E3 regulates neddylation that reported been has It c 2Xadta niiino edlto,b sn UBC12 using by neddylation, of inhibition that and H2AX ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal c 2Xuiutlto Fg D.Teubiquitin The 4D). (Fig. ubiquitylation H2AX c 2Xuiutlto pntetreatment the upon ubiquitylation H2AX c c c c 2XadBC1i el hthad that cells in BRCA1 and H2AX 2X(i.5) h xrsinof expression The 5C). (Fig. H2AX 2XadBC1a N damage DNA at BRCA1 and H2AX 2Xta edtce yusing by detected we that H2AX c 2XadBC1were BRCA1 and H2AX c 2Xare H2AX 2243

Journal of Cell Science EERHARTICLE RESEARCH aae hc togysgetdta edlto fet the DNA affects that 2244 to neddylation found response that the in suggested also strongly Ubc13 suppressed We which and NEDP1 VP16. damage, RNF168 and with decreased between NEDP1, interaction but interaction treatment with min The interacted 5 upon 6B). RNF168 within min (Fig. increased 30 Ubc13 Ubc13 the in with E2 in RNF168 and of reduction RNF168 an a in between resulted but 6A). (Fig. VP16 ubiquitylation, RNF168 with endogenous of the cells neddylation, of of treatment endogenous analysis upregulation the pulldown of His-tag that damage. neddylation DNA showed and to response ubiquitylation and/or in the RNF168 ubiquitin the investigated we RNF168 in repair, of damage changes DNA function in the conjugation NEDD8 elucidate further neddylation RNF168 To modulates repair damage DNA E3 ubiquitin its regulates RNF168 activity. of ligase neddylation that indi confirm the with transfected were cells HEK293T activity. ligase His– ubiquitin with E3 ubiquitylated 6 RNF168 transfected of impair were 50 quantification mutants cells without The HEK293T Ubc12 or investig activity. blotting. (D) also with ligase was blotting. 50 treated the ubiquitin neddylation without western and E3 with RNF168 or and plasmids with detected. transfected RNF168 down treated were abolishes were then pull RNF168 and cells NEDP1 without His-tag h HEK293T Deneddylase or activity. by 24 with (C) blotting for E3 ubiquitylation NEDP1. western HA–RNF168 its H2A or and of on impairs Flag–NEDP1 down absence NEDP1 ubiquitin, and pull or of His-tag RNF168 effects presence by to the the investigated and NEDD8 in then h, of were 24 conjugation RING2 for the or plasmids RNF168 abolishes indicated RNF8, NEDP1 of (B) ubiquitylation lysate. activity. and ligase whole-cell neddylation E3 the RNF168 plasmids, regulates indicated NEDD8 the of Conjugation 5. Fig. s.e.m., et eeaie h feto edlto nteinteraction the on neddylation of effect the examined we Next, n 5 3. m P6fr3 r6 i.Uiutlto fHAand H2A of Ubiquitylation min. 60 or 30 for VP16 M c 2Xa l ieonsi hw n a efre sdsrbdi i.1.* 1B. Fig. in described as performed was and shown is timepoints all at H2AX A N18i ohuiutltdadndyae.HK9Tclswr rnfce with transfected were cells HEK293T neddylated. and ubiquitylated both is RNF168 (A) m eosrt htndyainpasa motn oei the in role important we an 5, activity. RNF168 Fig. plays be of in be neddylation regulation presented to now that results remains can the still demonstrate with RNF168 residues of Combined lysine site determined. ubiquitylation these the that multiple substrates Although excluded, other on with neddylated. These modified consistent is are probably S3B). which is NEDD8, the of Fig. by RNF168 of conjugation lysines that that material the the to suggest on (supplementary comparison results of in effect protein RNF168 similar found to wild-type analysis a We NEDD8 and (ESPript). had and ubiquitin sequence mutations acid all (UbPred) amino that sites mutated the conjugation of were at of prediction 445 conservation the ubiquitin on and Lysines based 441 selected for S3B). 232, were which 227, arginine, Fig. 140, to 126, material 112, positions (supplementary constructs ubiquitin-conjugating E2 the and enzyme. RNF168 between interaction P6fr3 r6 i,uiutlto fHAand H2A of ubiquitylation min, 60 or 30 for VP16 M utemr,w eeae eiso N18mutation RNF168 of series a generated we Furthermore, ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal c 2Xwr netgtdb i-a uldw n western and down pull His-tag by investigated were H2AX P , .5 *** 0.05, c 2Xwsinvestigated was H2AX P , .0,means 0.001, WCL, . cated ated

Journal of Cell Science EERHARTICLE RESEARCH iae hra t neato ihteE nyeUc3i euae by regulated H2A. is modified Ubc13 of enzyme number E2 the E3 the +, to an with NEDP1. levels as interaction initial process its to antagonistic whereas increases this ligase, H2A regulates of ubiquitylation RNF168 neddylation repair. the the complete, terminate as and is such decreases process proteins, H2A repair repair the of of When recruitment Rap80. the and H2A facilitate BRCA1 damage of to DNA neddylation order When whereas in ratio. increases, decreases balanced H2A a of in ubiquitylation NEDD8 occurs, and ubiquitin repair. damage by DNA modified regulates Neddylation 7. Fig. neddylated and ubiquitylated both is model H2A the As 7, H2A. Fig. damage of neddylation DNA in the of by shown mechanism mediated is regulatory that negative repair H2A, a of neddylation and propose and we function ubiquitylation the the competitive growth, the between studied investigating relationship cell By we neddylation. in H2A Here, of mechanism functions development. regulatory and viability important plays immunoprecipitation. NEDD8 IP, the Flag. then and and treate HA and min, against DISCUSSION plasmids 30 indicated antibodies or the using Ned with 15 performed (B) transfected for lysate. was were VP16 whole-cell cells assay HEK293T WCL, without co-immunoprecipitation Ubc13. ubiquitin; or enzyme Ub, a with conjugating NEDD8; VP16, E2 N8, treated assay. without the and down and or pull plasmids RNF168 His-tag between indicated the interaction the following the with investigated regulates was transfected RNF168 RNF168. were of of cells modification neddylation HEK293T endogenous and damage. ubiquitylation DNA modulates after repair gradually damage DNA 6. Fig. nrsigcls 2 is H2A cells, resting In nrsigcls pnDAdmg,HAi predominantly is H2A damage, DNA Upon cells. resting in 2 bqiyain edmntae htRF6 sa E3 an is observation, our RNF168 to Similar neddylation. that H2A mediates demonstrated that ligase we ubiquitylation, spatially H2A and H2A. effect to steric ubiquitin a of possible has conjugation is NEDD8 the it blocks of acids, conjugation amino that 76 the have that and NEDD8 and lysines only ubiquitin contains mature arbitrary the H2A Because of on H2A. tail of C-terminal H2A ubiquitylation that inhibits to indicate conjugation data NEDD8 conjugated These H2A. is on not sites NEDD8 did multiple H2A to can conjugated of NEDD8 on that or be C-terminus suggesting mutation, site neddylation, the H2A that in abolish the revealed completely relationship residues neddylation lysine of for of competitive Investigation required truncation, is NEDD8. the that and H2A demonstrated ubiquitin between we expression, H2A by regulated that negatively conclude is to process neddylation. reasonable repair is damage it DNA ubiquitylation the therefore, H2A 4C); of (Fig. that of decreased whereas level the increased, repair, and neddylation facilitate damage DNA to H2A cells with We likely resting Along is repair. BRCA1. in which damage DNA occurred, H2A protein the damage to when repair conjugated removed H2A suppresses was was damage that NEDD8 DNA that and demonstrated found the we ubiquitylation of Here, recruitment 2009). antagonizes al., neddylation et and H2A Pinato of ubiquitin by chains damage 2012; their ubiquitin DNA of the through sites to to proteins, binding recruited are repair (UIM), DNA motif interacting many as process this Ubc13. RNF168 that enzyme of and interaction E2 the neddylated the and for with strongly of essential we also was neddylation increases conjugation mediates was Additionally, NEDD8 that RNF168 H2A level. ligase that E3 basal H2A, an of as the RNF168 neddylation of to identified stages decreases later repair, the ubiquitylation At damage order process. in repair reduced DNA subsequent is the protein facilitate the to of neddylation and ubiquitylated, neetnl,i diint ucinn sa 3lgs for ligase E3 an as functioning to addition in Interestingly, protein endogenous and ectopic both of conditions Under repair damage DNA the in event key a is ubiquitylation H2A A N18uiutlto nrae n N18ndyainreduces neddylation RNF168 and increases ubiquitylation RNF168 (A) ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal , 0aioais whereas acids, amino 10 c 2X(at tal., et (Gatti H2AX dylation 2245 with d

Journal of Cell Science bevto niae ht terysae ftersos to response This the that of S3A). stages found Fig. early and at material but that, VP16 5C) (supplementary indicates (Fig. selection with observation ubiquitylation neddylation substrate H2A treatment the not promotes upon strongly examined Vousden, p53 RNF168 We RNF168 and regulate Carter 2004). of 2007; modifications Harper, al., two ubiquitylation et 2008; p53 (Abida these both coordinately mediate and activity to ligase neddylation, E3 and an as acts MDM2 ARTICLE RESEARCH 2246 following neddylation whereas, RNF168 by RNF168, response of of regulated the and activity amount of was ligase certain H2A E3 stages process a the early damage, assures this the DNA At and to RNF168. treatment the the 6B), of after (Fig. that neddylation of min result VP16 30 the termination decreased with by interaction Upon reflected interaction RNF168–Ubc13 decreased, 6). the gradually (Fig. and RNF168 H2A that following VP16 of min observation 5 ubiquitylation within with our increased Ubc13 treatment 2007). by and al., et RNF168 supported Mailand between 2009; is al., et This (Doil response damage and of DNA H2A of neddylation ubiquitylation the of c activity RNF168-catalyzed the and ligase 6). E3 (Fig. RNF168 the with of reduces RNF168 further between interacts which NEDP1 Ubc13, interaction enzyme RNF168 E2 by the suppresses that inhibition RNF168 demonstrated and 2004; E3 we the NEDP1, the al., stabilizes Here, promotes and et enzymes, neddylation E2 Pan ligase. and that E3 2001; suggest the between al., 2004), interaction al., et as et (Kawakami neddylation such ligases, Xirodimas MDM2 E3 damage RNF168 other and of DNA neddylation its that cullin affects the on for further studies and The essential and repair. activity, ubiquitylation is H2A data RNF168 ligase our regulates directly of importantly, E3 RNF168 neddylation More 2013). of ubiquitin that al., UIM indicate et the is (Ma here which that NEDD8 neddylation, to finding of binds the substrate a with we al., itself Furthermore, et consistent 2009). is Mailand damage al., RNF168 2007; et DNA that Pinato al., found other 2012; et al., of (Huen et recruitment Mattiroli sites 2007; lesion the ubiquitylation at the for proteins that essential repair fact to is the with ubiquitin H2A agreement of conjugates in is preferentially which H2A, RNF168 damage, DNA atr ta. 08 aeaaie l,20;Wn tal., et Wang 2006; repair. al., damage (Guerrero- DNA et VP16-induced repair in Kapetanaki reported not damage 2008; are Tanaka, but neddylation al., 2006) Cul4B DNA and and et are Cul4A (Chiba UV-induced 2009). Santoro al., studied Cullins in et Huang well 2008; participate 2011). al., been et Duda al., have 2004; in that a et involved that are substrates (Liao indicate proteins response analyses damage neddylation Proteomic DNA of of UIMs 2013). also the number al., by large Neddylation recognized et to be (Ma response. could reported that of RNF168 repair sites deprivation been lesion DNA on and occurred the has sites, damage impairs RNF111 DNA RNF111 at ligase neddylation E3 catalyze The complicated. to antagonizes order also protein. VP16 in the RNF168 of necessary of after ubiquitylation neddylation are the min the 30 studies whether noted Further RNF168, elucidate also 6A). of by (Fig. We accompanied ubiquitylation treatment was Ubc13. RNF168 increased enzyme The of the neddylation E2 6A). decreased (Fig. its the interaction that and VP16 the impaired RNF168 with further between RNF168 treatment of after neddylation reduced min 30 decreased 2Xi eyerysga,wihocr ihn1 i,i the in min, 10 within occurs which signal, early very a is H2AX h oeo edlto nteDAdmg earntokis network repair damage DNA the in neddylation of role The c 2Xuiutlto,ndyaino RNF168 of neddylation ubiquitylation, H2AX c 2X h 3lgs ciiyof activity ligase E3 the H2AX, etr.ND1 B1-11 n B1-11 eecoe into pRK7-HA cloned and were pEF1-C-6His UBC12-C111A into and pCDNA human UBC12-C111S cloned encoding NEDP1, the were cDNA vectors. The into NEDD8 (3Flag). and cloned repeats ubiquitin Flag-tag were three mutants K119R/K120R/K126R/K128R/K130R with vector H2A-1-118 and (H2A-4KR), (H2A-5KR); K119R/K120R/ (H2A-3KR), K126R/K128R K119R/K120R/K126R H2A-K119R/ (H2A-2KR), H2A-K120R, H2A-K119R, K120 mutants H2A the H2A; Human Plasmids METHODS AND MATERIALS fhsoe sflos el eeclue n1-mdse and dishes guanidinium-HCl, M (6 10-cm buffer lysis in cells of transfection, Na ml cultured after 6 M h in 0.1 were lysed 48 and At Cells harvested plasmids. were indicated follows. the modification with as the transfected examine to histones performed was of assay pulldown His-tag The assay pulldown His-tag for serum) bovine 37 fetal at 10% with h supplemented 24 Dulbecco’s been Modified had Iscove’s (that in cultured Medium were cells HeLa and HEK293T transfection and culture Cell rabbit (E1383) a VP16 Pharmaceuticals. Bioworlde, Origene. Sigma-Aldrich. Millennium from from from from was monoclonal gift was a were (TA306771) rabbit was RNF168 MLN4924 (BS1036) against Motif, antibody BRCA1 Active monoclonal and from (BS1662) was against antibody (39209) antibodies monoclonal rabbit H2A a and Medical against Japan), from were (Nagoya, (PM053) Laboratories actin mouse against Biological (D291- monoclonal from tag rabbit companies: were His a the and (H9658) 3) against following HA antibodies or monoclonal the (F3165) mouse Sigma-Aldrich, Flag from against antibodies purchased monoclonal were Antibodies reagents and Antibodies vector. pCDNA-3Flag the ufrI( undnu-C,01MNa M 0.1 guanidinium-HCl, M (6 I buffer ecpotao) ufrII( ra . Na M 0.1 urea, M (8 III buffer mercaptoethanol), n 0mM 10 and lsisuigMgTa . rnfcinraet(rgn)acrigto instructions. according (Origene) manufacturer’s reagent the transfection 1.0 MegaTran using plasmids C,p . n 0mM 10 and 8.0 pH HCl, rtnX10 n ufrI 8Mue,01MNa M 0.1 urea, mM 10 M 30 6.3, (8 X-100). pH mM IV Tris-HCl, 10 buffer M and 0.01 and 6.3 X-100) pH Triton Tris-HCl, M 0.01 idnssgetanwyietfe euaoymcaimfor and pathway. the mechanism neddylation regulatory activity the through of identified ligase repair newly damage Downregulation DNA E3 a suggest its activity. and findings H2A impairs of ligase ubiquitylation RNF168 H2A inhibits ubiquitin of of neddylation promotes neddylation E3 RNF168 damage of neddylation hand, DNA the its hand, the other one the suppresses on and response; the ubiquitylation This its On antagonizes response. S4). investigation. VP16-induced damage further in require Cul4A will Fig. which for repair role damage a material DNA indicates H2A result on (supplementary unexpected Cul4B H2A suppressed strongly and Cul4A Surprisingly, ubiquitylation whereas Cul4A VP16. effect, with little of had treatment Cul4B to effect response the in ubiquitylation examined thus We sn P4 yi ufr eeue sipt.Telst a incubated was lysate The inputs. as used 70 were with buffer, lysis NP-40 using Na ape,wt neulvlm f2 of volume equal an with samples, ySSPG n etr ltanalysis. blot western and SDS-PAGE by ee ecnimterl fpoenndyaini h DNA the in neddylation protein of role the confirm we Here, 2 HPO ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal m 4 2 lofNi HPO ˚ m NaH CO 5% under C b feuinbfe a sdt lt oiidpoen.The proteins. modified elute to used was buffer elution of l mratehnl o 0mn %o h el,lsdby lysed cells, the of 2% min, 30 for -mercaptoethanol) 4 2 2+ PO NaH c ed o n hnwse ortmswt ml 1 with times four washed then and h 4 for beads 2X(S70,HB(S67,H B17) H4 (BS1174), H3 (BS1657), H2B (BS4760), H2AX 4 .1MTi-C,p . n 0mM 10 and 8.0 pH Tris-HCl, M 0.01 , 2 PO b 4 mratehnl,bfe I( ra . M 0.1 urea, M (8 II buffer -mercaptoethanol), .1MTi-C,p .,5m imidazole mM 5 8.0, pH Tris-HCl, M 0.01 , 2 n hntasetdwt h indicated the with transfected then and 6 b mratehnlpu .%Triton 0.1% plus -mercaptoethanol D odn ufr eeanalyzed were buffer, loading SDS c 2X ae oehr our together, Taken H2AX. 2 b HPO mratehnlpu 0.2% plus -mercaptoethanol 4 NaH 2 2 2 PO HPO HPO 4 .1MTris- M 0.01 , 4 4 NaH NaH 2 2 PO PO b 4 4 - , ,

Journal of Cell Science osdrdsaitclysignificant. statistically considered au a eemndb Student’s by determined was value ape eeaaye ySSPG n etr blotting. western and SDS-PAGE by analyzed were Samples n hnwse ihpecildPStretms ial,20 Finally, times. three PBS pre-chilled with washed then and al MET,1 P4,p .)wt 5 with 7.4) 5 40 PBS pH with pre-cleared mM then pre-chilled NP-40, were 150 lysates Tris-HCl, of 1% The mM cocktail. ml EDTA, (50 buffer 10 lysis mM NP-40 indicated with 1 ml washed the 1 NaCl, in were min with cells 60 transfected for later, lysed and and h dishes 48 10-cm At plasmids. in cultured were Cells Co-immunoprecipitation ARTICLE RESEARCH bd,W . ioav . ho . hn,W n u W. Gu, and W. Zhang, W., Zhao, A., Nikolaev, M., W. Abida, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.138891/-/DC1 online available material Supplementary material Supplementary and CRP/CHN09-01]. Engineering number number Genetic [grant [grant for China Centre Biotechnology of International Education the of and number Ministry 20130001130003]; [grant of programs Fund Technology 973 Doctoral High China 2010CB911800]; of National of Foundation the Program Science 31170709]; Development National and and the 30930020 from numbers grants [grant by China supported was work This Funding and Z.J.H. experiments. the experiments. of some most performed performed X.H. J.H.G. prepared and and T.T.L. data manuscript. interpreted the experiments, the designed X.F.Z. and J.H.G. T.T.L., contributions Author interests. competing no declare authors The interests Competing providing for Pharmaceuticals Millennium thank OH) University, We MLN4924. State construct. Ohio Cul4A (The the for Zhu Qianzheng for China) and and University, RNF8 plasmid (Zhejiang Ubc13 of Zhu the plasmids Yongqun providing of thank gift for sincerely Centre the We and for RNF168. Biology Denmark) Cancer of Research, (Institute Stress Lukas Genotoxic Claudia and Lukas Jiri thank We Acknowledgements SPSS means using as represented performed are were Data 20). procedures (version variance statistical of software analysis All one-way using by (ANOVA). analyzed were data quantified The analysis Data at min with 10 fixed then for and methanol later, PBS pre-chilled h pre-chilled 24 of with plates. ml times 6-well 1 three in washed coverslips glass were on cells cultured were cells HeLa microscopy Immunofluorescence ahn ihpecildPStretms h el eeicbtdfor incubated were cells the 37 times, at three h 1 PBS pre-chilled with washing nioista a endltdi %BAfr1ha 37 at h 1 for primary BSA with 1% incubation bovine in by 1% diluted followed with been h, blocked had 1 were that for cells antibodies (BSA) the albumin times, serum three PBS pre-chilled ial,tepoenGwswse ih1m fN-0lssbfe three buffer lysis 96 NP-40 at of min ml 10 for 1 heated with then washed and was times G protein the Finally, atr .adVudn .H. K. Vousden, and S. Carter, onigslto a sdt on el.Alo h mgswere images the of (CarlZeiss, microscope All DuoScan and cells. NLO 710 mount Germany). LSM to a using used by captured was solution mounting vrih t4 at overnight hb,T n aaa K. Tanaka, and T. Chiba, eBe . aro-ee,D n icaoe,A. Ciechanover, and D. Zaaroor-Regev, P., Bie, de m rmtsteNdyaino 5 n niisistasrpinlactivity. transcriptional its inhibits and p53 Chem. of Neddylation the promotes bqiiain uolto n edlto fp53. of neddylation and sumoylation ubiquitination, ED-ojgtn system. NEDD8-conjugating oyobpoenRN1 bqiiainb USP7. by ubiquitination Commun. RING1B protein Polycomb fatbd rIGwsue obn otebi rtisfr5h or h, 5 for proteins bait the to bind to used was IgG or antibody of g 282 ˚ ihscnayatbde hthdbe iue n1 BSA 1% in diluted been had that antibodies secondary with C 400 1797-1804. , ˚ ,adte nuae ih80 with incubated then and C, 389-395. , 20) ulnbsduiutnlgs n t oto by control its and ligase ubiquitin Cullin-based (2004). ur rti et Sci. Pept. Protein Curr. 20) 5-b uin smdl of models as fusions p53-Ubl (2008). ˚ t ih30 with C etadavleof value a and test m 2 rti o ute h. 3 further a for G protein l 20 m elCycle Cell m ˚ l2 m 21) euaino the of Regulation (2010). 5 .Atrwsigwith washing After C. ice.Bohs Res. Biophys. Biochem. fpoenG Usually, G. protein of l fpoes inhibitor protease of l 177-184. , 6 D odn buffer. loading SDS 6 20) FBXO11 (2007). 7 ...The s.e.m. 2519-2528. , P , ˚ .5was 0.05 .After C. .Biol. J. m lof P a . hn . hn,F,Yn,C . ag .adY,X. Yu, and S. Wang, Y., C. and Yang, K. F., Zhang, Garcia, Y., H., Chen, T., Bernard, Ma, C., D. Bouck, L., J. Blank, J., X. Liu, H., Liao, Rapic L., C. Hsieh, C., D. Bisi, J., Guerrero-Santoro, G., M. Kapetanaki, T. E. Yeh, and P. H. Nguyen, K., Kito, Huang, T., and Kamitani, Q. Z. Pan, N., Nillegoda, C., Guerrero, Y., Yang, K., Wu, J., Jones, P. E. Candido, and D. Jones, O. M. J. Chen, Dayhoff, and and B. T. M. Yaffe, L. X., Hunt, Yu, K., Minn, I., Manke, R., Grant, S., M. Huen, aln,N,Bke-esn . asrp . eadr . atk . Lukas, J., Bartek, F., Melander, H., Faustrup, S., Bekker-Jensen, N., Mailand, ikpuo,D,Degs . aucesi .adJnsh S. Jentsch, and K. Matuschewski, G., Doenges, Chock, D., and Liakopoulos, C. D. Yang, G., Wang, E., Tekle, F., R. Shen, R., Santockyte, M. T., Goebl, Li, J., Callis, Y., Liu, W., Jiang, M., J. Laplaza, N., Mathias, D., Lammer, M. Hochstrasser, and R. Felberbaum, O., Kerscher, Suzuki, N., Minato, K., Yamanaka, K., Iwai, T., Suzuki, T., Chiba, T., Kawakami, Scott, M., D. Duda, M., A. Taherbhoy, W., H. Hunt, O., Ayrault, T., D. Huang, ares .N,E esod-uet . lekn . im .adPeters, and K. Hiom, S., Elderkin, S., Messaoudi-Aubert, El N., G. Maertens, apr .W. J. Harper, A., R. Greenberg, A., Datta, V., Gupta, J., Zhu, E., C. Berndsen, I., M., Gorbachinsky, C. Guzzo, L., C. Hsieh, G., M. O., Kapetanaki, M. Olson, J., C., Guerrero-Santoro, L. Yeoman, M., R. Baum, W., L. C. Taylor, Penengo, L., and I. S. Polo, Goldknopf, P., Soffientini, E., Maspero, S., Pinato, M., Gatti, jre . hms . hn . el,A,Cra,S,Spr,N,Dc,L R. L. M. Dick, Hochstrasser, N., Shpiro, S., Curran, A., Zemla, J., Chen, Y., Thomas, R., Hjerpe, Y. Zhang, and E. Li, B., Chadwick, T., Schulman, Chen, and J., M. Fang, Hammel, W., H. Hunt, C., D. Scott, A., L. Borg, M., D. Duda, Pepperkok, H., D. Larsen, P., Menard, S., Bekker-Jensen, N., Mailand, C., M. Doil, Dobbelstein, and M. Koeppel, C., Dohmesen, ouainuo niiino h ED-ciaigezm yMLN4924. by enzyme NEDD8-activating Proteomics the Cell of inhibition upon S. modulation E. Lightcap, 2208-2214. protein. 272 ubiquitin-like down-regulated developmentally a NEDD8, terminal proteins. associated and embryogenesis L. for hypodermis. required the of is differentiation A24. elegans protein Caenorhabditis nuclear of component Commun. nonhistone Res. Biophys. the and ubiquitin and ubiquitylation histone assembly. via protein signal checkpoint DNA-damage the transduces RNF8 (2007). rasadpooe sebyo earproteins. repair of assembly promotes and J. breaks Lukas, and C. eedn edlto ciae N aaeidcdubiquitination. damage-induced DNA activates neddylation dependent system. ubiquitin the to related pathway modification protein novel modifiers. ubiquitin-like for substrates B. P. complex. SCFCdc4 the of function affects rub1p protein M. Estelle, and proteins. ubiquitin-like 159-180. and ubiquitin by al. proteins et F. ligase. Osaka, E3 Y., SCF to Hidaka, ubiquitin N., UV- Shimbara, at H2A H., histone targets and E sites. group DNA S. pigmentosum damaged xeroderma A. in Levine, deficient and V. al. cascad et F. NEDD8 M. Roussel, the J., P. of Murray, modification. G., expansion Neale, A., RING L. Borg, C., D. fteIKatmu suppressor. tumour INK4a the of G. 49 bqii hisaesgasfrRP0adteeymdaetercutetof recruitment the damage. mediate DNA thereby of and sites RAP80 to for J. BRCA1 signals M. are chains Matunis, ubiquitin and C. Wolberger, ubiquitinates and chromatin UV-damaged to H2A. binds histone ligase Rapic protein and DNA-binding S. A. Levine, protein. chromosomal H. non-histone 250 ‘‘histone-like’’ Busch, a and A24, protein W. A. by Prestayko, targeted H2As histone of tail N-terminal ligase. the ubiquitin at RNF168 mark ubiquitin novel A (2012). ocl yl regulation. cycle cell to enzymes. ubiquitin by NEDDylation T. Kurz, and p53. to Nedd8 in involved is and inactivation. chromosomes X X of inactive initiation with associates ubiquitination H2A conjugation. of control conformational allow A. to B. chromosomes damaged al. proteins. on et repair conjugates J. of Bartek, ubiquitin accumulation D., amplifies Durocher, and S., binds Panier, J., Ellenberg, R., Tip60. by neddylation Mdm2-mediated 897-907. , 20) agtdpoemcaayi fteuiutnlk oiirnd8and nedd8 modifier ubiquitin-like the of analysis proteomic targeted A (2008). 21) bqii-pcfcpoess7ad1 ouaePlcm regulation Polycomb modulate 11 and 7 proteases Ubiquitin-specific (2010). 28557-28562. , 7182-7187. , 20) tutrlisgt noND8atvto fcli-IGligases: cullin-RING of activation NEDD8 into insights Structural (2008). 20) eea prahfrivsiaigezmtcptwy and pathways enzymatic investigating for approach general A (2006). ora fCl cec 21)17 2824 doi:10.1242/jcs.138891 2238–2248 127, (2014) Science Cell of Journal 20) edltn h urin d2ctlzdcnuainof conjugation catalyzed Mdm2 guardian; the Neddylating (2004). o.Cell Mol. acrRes. 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