Journal of Cell Science RA seby nelnn h motneo hs idns eso htSAC5dpeinrnesclssniiet Rand SMARCA5- IR DSBs. poly(ADP-ribosyl)ation, to of damage-induced sensitive repair DNA cells and and between renders link conjugation signaling depletion functional RNF168-dependent ubiquitin SMARCA5 a and that efficient unveils remodeling show study facilitates chromatin Our we subsequently mediated defects. findings, repair which these DSB DSBs, of in importance at results RNF168 the accumulation manner. Underlining binding PARP-dependent RNF168 assembly. SMARCA5 and promoting BRCA1 for damage- required by DNA were a response chains in DNA ubiquitin poly(ADP-ribose) interact to and RNF168 response RNF168 vivo and ubiquitin while SMARCA5 RNF168-driven in damage, throughout that the DNA complex show of after RAP80–BRCA1 we organization poly(ADP-ribosyl)ated this, the spatial became of the and support regulates RAD18 PARP In factors that damage. ubiquitin-binding suggests the This and chromatin. conjugates DSB-flanking ubiquitin RNF168, ligase ubiquitin o:10.1242/jcs.109413 doi: 889–903 126, Science Cell of Journal 2012 December 3 Accepted ihlsSroczynski Nicholas MRA/N2 oRF6-eedn N damage signaling DNA RNF168-dependent to remodeler SMARCA5/SNF2H chromatin the links Poly(ADP-ribosyl)ation Article Research N-Kmdae hshrlto fhsoeHA (termed H2AX ATM/ the histone is c formation of IRIF in phosphorylation step key DNA-PK-mediated A 2010). Chen, 2010; ionizing and Mailand, Huen as and (Bekker-Jensen known (IRIF) foci structures radiation-induced discernible 2006). and cytologically Kanaar, forming and (Jackson Wyman 2010; (HR) al., et recombination Negrini 2009; homologous Bartek, end-joining cell non-homologous and involves in as repair delay (NHEJ) known DSB as pathways 2010). well dedicated Chen, as two and lesions, (Huen these To progression the of cycle 2010). lead repair activate that and al., cells events detection comprises DSBs, to et which (DDR), of Negrini response damage effects DNA 2009; deleterious Bartek, the cell circumvent and or development (Jackson cancer chromosome in to death result stability. lead may can genome which DSBs to of rearrangements, threat repair arise major inaccurate or or a (IR) Inefficient are radiation replication, ionizing to DNA cells during be of can exposure which by (DSBs), generated breaks double-strand DNA Chromosomal Introduction words: Key although remodeling of activity, chromatin PARP ISWI spreading context Remarkably, of the manner. efficient (PARP1)-dependent subunit RNF8/RNF168-dependent in 1 catalytic the regulated polymerase an the is poly(ADP-ribose) for SMARCA5/SNF2H, response elicit a in that dispensable this chromatin DSBs show how to we Precisely native recruited Here, factors. is in unexplored. complexes, repair largely various arising remains of (DSBs) chromatin recruitment of the breaks triggers double-strand which response, DNA ubiquitylation (IR)-induced radiation Ionizing Summary work this to equally ` contributed authors *These 3 2 Attikum 1 van Haico and Smeenk Godelieve uhrfrcrepnec ( correspondence for Author ooNrikFudto etrfrPoenRsac,Fclyo elhSine,Uiest fCpnae,20 oehgn Denmark Copenhagen, 2200 Copenhagen, of University Netherlands Sciences, The Health Rotterdam, of GE, Faculty 3015 Netherlands Research, Center, The Protein Medical Leiden, for Erasmus ZC, Center Genetics, 2333 Foundation Biomedical 20, Nordisk for Einthovenweg Novo Center Center, Genetics, Medical of University Department Leiden Toxicogenetics, of Department 2X.MC usqetybinds subsequently MDC1 H2AX). 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. ueosDRpoen cuuaea RidcdDSBs, IR-induced at accumulate proteins DDR Numerous xliighwSAC5i ikdt h N18uiutncsae oevr MRA a on orglt the regulate to found was SMARCA5 Moreover, cascade. ubiquitin RNF168 the to linked is SMARCA5 how explaining , MRA/N2,Crmtnrmdln,Pl(D-ioe oyeae AP N18 N aaesgaigadrepair and signaling damage DNA RNF168, PARP, polymerase, Poly(ADP-ribose) remodeling, Chromatin SMARCA5/SNF2H, [email protected]) 1, ,Wue .Wiegant W. Wouter *, 3 hmsCostelloe Thomas , 1, ` c 2Xadmediates and H2AX c 2Xit aae hoai,slcieypooe pedn fSAC5 h E3 the SMARCA5, of spreading promotes selectively chromatin, damaged into H2AX 1 1, o .Romeijn J. Ron , ,Jre .Marteijn A. Jurgen *, ean agl lsv.Rcn tde aebgnt xlr this van explore to accomplished begun 2012a; is have DSB-flanking studies this al., the Recent how elusive. et largely that However, in remains 2009). (Luijsterburg clear Gasser, DDR modifications and becoming Attikum the is histone controls it chromatin and between in damage modified crosstalk extensively DNA non-histone are to and Histones histone response chromatin. through into DNA, proteins, to genomic of response, factors packaging response ubiquitin 2012). DSB al., RNF168-driven et of (Gudjonsson chromosomes the also spreading undamaged studies excessive of Recent UBR5, preventing 2007). and antagonists TRIP12 al., 2007; as al., such et as in et ligases, Wang Mailand ubiquitin 2007; several 2009; al., culminating al., implicated al., et et Huen et (Bekker-Jensen 53BP1, 2009; Stewart al., repair et and several DNA Doil 2010; and RNF8/ BRCA1 of activation by checkpoint including recruitment catalyzed RNF168 proteins, ubiquitylation promotes UBC13. and conjugase chromatin ubiquitin RNF8 RNF168/HERC2/UBC13 E2 ensuing ligases the and ubiquitin The HERC2 E3 with the together of recruitment 1 fiin inln n earo Ssi aprdb the by hampered is DSBs of repair and signaling Efficient letPastink Albert , 2 atj .Luijsterburg S. Martijn , 1 il Mailand Niels , 1 , 3 i Vermeulen Wim , 889 2 Journal of Cell Science 890 bevdi oto el t6h(upeetr aeilFig. material repair, (supplementary levels DSB of h to rate 6 the returned attenuates at loss nearly SMARCA5 cells Thus, but S1C,D). control cells, levels in control SMARCA5-knockdown observed DSB in to IR Conversely, compared after (supplementary timepoint). h cells 2 h cells higher 0 spontaneous significantly were control S1C,D, Fig. prevent and material cells in levels may DSB SMARCA5-depleted similar induced treatment IR S1C,D, Importantly, SMARCA5 samples). Fig. were material cells, (supplementary levels breakage SMARCA5-depleted that chromosome DSB non-irradiated assay. DSB comet in suggesting IR-induced neutral elevated in the SMARCA5 slightly used of we involvement rejoining, NHEJ the by assess repair DSB To for required Fig. is material SMARCA5 supplementary 1A,B; (Fig. ATM depletion depletion S1A,B). or as SMARCA5 extent XRCC4 same al., RNAi-mediated the et of to (Lan sensitivity that study IR increased previous found markedly a we with agreement 2010), In after IR. cells to mammalian of exposure survival on clonogenic Based affects response. knockdown DSB in we the study findings this in In SMARCA5 the 2000). of al., et role Poot the 2000; examined al., Ito et 2002; chromatin LeRoy al., 1999; et ATP-dependent Shen, al., Collins et 2003; and of al., (Bao et subunits (Barak family catalytic complexes remodeling ATPase are SNF2 and the 2007), of subclass ISWI the in screen IR RNAi genome-wide against A cells human protects SMARCA5 Results RNF168- the DDR. and the in process poly(ADP-ribosyl)ation, signaling remodeling dependent between chromatin link functional SMARCA5-mediated we novel Here SMARCA5 limited. how currently a is DSBs into uncover of insight repair DSB and mechanistic signaling the the However, affects Sa 2011; in 2011). al., ISWI al., et involved Nakamura distinct et 2010; also al., several et is (Lan of response complexes, require ATPase remodeling also the chromatin BRCA1, found was SMARCA5/SNF2H, it or optimally Recently more that known. RNF168 not whether to is as process activities However, chromatin-modifying this such possibly 2011). of factors, Attikum, step response, downstream the van each DSB for and at structure (Luijsterburg the activities chromatin the of remodeling reconfigure p400 chromatin stages that response specific various shown DSB distinct was recruit while that 2010), It suggesting al., factors RNF8, et 2010). by (Xu recruited al., MDC1 is 2012a; by CHD4 et regulated al., is Xu et DSBs to Luijsterburg 2010; recruitment 2010; al., al., et et Smeenk (Larsen only and DSBs ubiquitin RNF8/RNF168 at not TIP60 the promote response the respectively, also but of repair, complexes, DSB subunits al., facilitate remodeling et catalytic at Park by chromatin the 2006; CHD4, al., phosphorylation NuRD repair et and Murr H2AX p400 2010; DSB al., 2006). and et (Lee facilitate respectively acetylation DSBs, histone complexes promoting remodeling SWI/SNF and TIP60 chromatin the that demonstrated and cells mammalian in 06.Dtbs erhsrvae w ua proteins, and highly similarity) similarity), human 79% 79% to two and homologous and identity revealed (64% identity SMARCA5/SNF2H searches (63% SMARCA1/SNF2L genes Database novel as 2006). well as factors, as DDR such known including genes, 45 isw-1 ora fCl cec 2 (4) 126 Science Cell of Journal hc rtc om gis R(a afe tal., et Haaften (van IR against worms protect which , .elegans C. .elegans C. efrttse hte SMARCA5 whether tested first we , S-.Bt rtisaemmesof members are proteins Both ISW-1. anradtselegans Caenorhabditis ´ ce -Molina nchez identified 2 IR u eut togysgetta MRA rmtsDBrepair DSB NHEJ. promotes SMARCA5 by 1C). that (Fig. suggest strongly results Hence, S2D). changes our Fig. fluorescence cycle material cell (supplementary of efficiency effect were NHEJ any the profiles GFP on out ruling cycle cells, Cell these in 1C). by altered same (Fig. not the knockdown XRCC4 to measured as NHEJ extent impaired factor as depletion NHEJ efficiency SMARCA5 the Interestingly, the reduced NHEJ BRCA2, of factor loss HR of the expected, of As not but S2A) 2008). 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S2B,C) SMARCA5 1D; Remarkably, al., Fig. (Fig. efficiency material et HR supplementary reduced (Weinstock dramatically depletion S2B) BRCA2 functional Fig. a material produces conversion olwn I-S Following speeti n ftodfcieGPrpre ee.Following genes. reporter GFP defective two I-S of one in present is h J-F eotrsse,wihmaue earbtentwo between I-S repair measures tandem which system, reporter EJ5-GFP the that Indeed, arrest suggest repair. this results progression. DSB from These promotes recover SMARCA5 S1E,F). cycle to Fig. failed cells, material cell control IR, (supplementary to after affect contrast phase in G2 but, the may in accumulated turn cells SMARCA5-depleted in which h RGPrpre ytm nti ytm igeI-S single using a by system, HR this in In SMARCA5 system. of reporter role DR-GFP possible the HR a by assessed repair we DSB Next for required is SMARCA5 CI)culdt uniaiePR(PR ofre these confirmed A (qPCR) flanking sites and different PCR SMARCA5 showing quantitative by 2011; results to al., et coupled Nakamura 2010; (ChIP) al., et Lan 2010; Sa al., et damage DNA (Erdel to sites recruited is SMARCA5 that DSBs reported work to Recent recruited rapidly is SMARCA5 hoai oprmn o tlat2hus(i.2A,B; (Fig. hours 2 least DSB-containing at the for in detected compartment be could chromatin SMARCA5 bound ecigmxmmlvl rud35mnts( minutes 3–5 accumulated, around rapidly levels DNA accumulate, SMARCA5-GFP maximum create 2A). primarily reaching not (Fig. settings did breaks laser applied strand XPA, the protein that suggesting repair excision nucleotide by in marked accumulates chromatin region entire SMARCA5-GFP the spanned and that tracks 2010). laser-induced found multiphoton al., also et we (Iacovoni S3A–D) Moreover, Fig. material (supplementary 22 ´ ce-oiae l,21) hoai immunoprecipitation Chromatin 2011). al., et nchez-Molina oass osberl fSAC5i HJ eemployed we NHEJ, in SMARCA5 of role possible a assess To ce xrsin earo h I-S the of repair expression, 1 ce ie htsprt a separate that sites I ce xrsin earo h w I-S two the of repair expression, I c 2Xo A5 Fg F.Ntby the Notably, 1F). (Fig. RAD51 or H2AX si IidcdDB ncrmsm and 1 chromosome on DSBs SI-induced GFP c GFP 2Xacmlto ttwo at accumulation H2AX ce -nue S ygene by DSB I-induced eefo t promoter. its from gene GFP ee(supplementary gene oigsequence coding t 1/2 5 ce 0s,and s), 40 I-induced ce site 1 Journal of Cell Science euaeSAC5asml tDAsrn ras Since factors breaks. other strand DNA whether at unclear assembly however, ubiquitin SMARCA5 E3 remains, regulate RNF20 al., et It (Nakamura the DSBs to for 2011). SMARCA5 of role recruitment the a in ligase implicated DSBs work to recruitment Previous SMARCA5 to contributes of PARP recruitment the 2A,B). while (Fig. slower GFP-MDC1, considerably is to 53BP1 kinetics similar damage show with DNA studies laser-inflicted at kinetic accumulates Finally, SMARCA5-GFP that S3E). Fig. material supplementary MRA/N2 euae N aaesgaig891 signaling damage DNA regulates SMARCA5/SNF2H tadbek n aaye h taheto A hison chains PAR of attachment the accumulation catalyzes and SMARCA5 breaks affect strand not S4A–D). Fig. did material supplementary 2C,D; it (Fig. laser-tracks, in lhuhAMadDAP niiinpeetdrobust prevented DSBs-tracks. inhibition at DNA/PK assembly and c van ATM SMARCA5 2011; Although chemical Attikum, impacts their whether van examined inhibition we and 2009), Gasser, Luijsterburg (Jackson and response Attikum 2009; DSB the Bartek, in roles and key play DNA-PK and ATM 2XII omto n reduced and formation IRIF H2AX eetyi a hw htPR1 hc id oDNA to binds which PARP1, that shown was it Recently rae ihPRi ( PARPi. with treated A5.Saebr 10 bar: Scale RAD51. were for cells immunostained min 15 After irradiation. laser multiphoton to subjected were SMARCA5–GFP expressing ( S2C. Fig. and material B supplementary in shown is siRNAs indicated the for efficiency Knockdown used. were HR DR–GFP the reporter containing cells that except ( shown. is experiments ausaemeans are Values 8B. Fig. and material B supplementary in shown is siRNAs indicated the for efficiency Knockdown (pCBASce). vector expression rnfce iha I-S an later with h transfected 48 and siRNAs the indicated with transfected were EJ5– GFP reporter NHEJ the containing loading ( a control. as A. used in was cells Tubulin of (WCE) extracts whole cell using analysis blot by western monitored were levels XRCC4 ( experiments. are means Values survival. scored clonogenic and for IR to exposed siRNAs, indicated the with transfected Sst rmt N repair. ( DNA promote to to recruited DSBs is and IR cells against protects SMARCA5 1. Fig. E A si ,ecp htclswere cells that except A, in As ) H0S4 el were cells VH10-SV40 ) 6 E23 cells HEK293T C) c ...o he independent three of s.e.m. 2Xaccumulation H2AX B MRA and SMARCA5 ) 6 c 2Xand H2AX ...o three of s.e.m. F ce m 2Scells U2OS ) D)AsinC, m. I Journal of Cell Science 892 AP Fg EF.Teerslsipiaetecatalytic the implicate with results treatment These as 2E,F). levels (Fig. similar PARPi to reduced Fig. PARP2, recruitment not material but SMARCA5 PARP1, supplementary of 2C,D; depletion Moreover, (Fig. S5A). cells PARPi in with abrogated was treated not accumulation but ALC1 reduced, whereas the significantly abolished, in was SMARCA5 DSB-tracks Interestingly, enzymes findings in DSBs. PARP accumulation These to of SMARCA5 2010). involvement 2009; of the al., al., recruitment test et et to Gottschalk Polo DSBs us 2010; 2012a; prompted the al., to al., et of CHD4 et Chou Luijsterburg recruitment 2009; and al., the ALC1 et (Ahel directs enzymes proteins, remodeling chromatin acceptor and itself ora fCl cec 2 (4) 126 Science Cell of Journal ciiyo AP nSAC5rcutett N strand DNA to recruitment SMARCA5 in breaks. PARP1 of activity N aaeidcdb ae ir-raito rgeslocal triggers micro-irradiation expansion laser the by chromatin inhibited. into induced is spread damage PARP to the DNA when fails in compartment SMARCA5 chromatin that reduction 2C; DSB-flanking suggests (Fig. a This laser-tracks noticed 3C). 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Journal of Cell Science Cer–N8admhryRF6 pedn nclsfo .Tetakwdhfrteepoen a omlzdt hto AF-2 ntesm el for cells same the in PAGFP-H2A of that 5 to bar: normalized were Scale was Cells channels. proteins track. two these in same for recorded width the conditions. track were in indicated The images RNF168 the E. and or from of laser RNF8 cells each multiphoton in mCherry-tagged the spreading of with mCherry-RNF168 spreading irradiated and and mCherry–RNF8 PARPi, expansion with chromatin treated monitor or simultaneously untreated to mCherry-RNF168 or RNF8 fe 8 h it ftergo hwn cuuaino h niae rtiswsmaue odtrieteices ntakwdh Individual width. track in increase the determine to measured was proteins indicated indicated the the of of accumulation immunostained expansion showing or the region proteins on the GFP-fusion inhibition of ( indicated PARP width measurements the of the of effect s expansion the 180 the 10 illustrating After for bar: chromatin images Scale photoactivated analyzed Representative colors shown. the and utilizes compartment. are of images irradiation damaged proteins expansion the laser the the to multi-photon throughout of next to Quantification proteins shown (white). subjected 5 endogenous table 256 bar: then look-up to Scale The PARPi, (black) images. image. with ATP-depletion were 0 the grayscale images treated or from to the grayscale ranging mock next 8-bit in intensities recorded shown in intensities pixel The is pixel cultured representing times. compartment on were indicated white based the Cells to table at black damage. look-up recorded from DNA were a ranging Images using inflict photoactivation. images and to colored PAGFP prior into formaldehyde photoactivate converted 4% simultaneously in fixed to chemically laser or multiphoton medium, a with irradiated locally xaso noreprmna e-p eepesdhsoeH2A histone expressed chromatin we of set-up, extent experimental this the our measure in whether to expansion order and throughout In factors response sites chromatin. 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PARPi (Fig. defective that BRCA1 endogenous the GFP-RAP80 found GFP-RAD18, and with of we expansion Consistent the conjugates, reduced 2007). significantly ubiquitin al., Sobhian et of 2007; al., Wang spreading et 2007; Kim al., 2009; al., et et RAP80– (Huang mediate the complex and RNF168 BRCA1 RAD18 3C,D). and factors (Fig. ubiquitin-binding RNF8 of inhibition by recruitment generated PARP conjugates after GFP-UBC13 was Ubiquitin impaired chromatin of S5B). DSB-containing significantly distribution along also Fig. the ubiquitin as conjugated that material extent and found same supplementary we the to Consequently, 3C,D; impaired pronounced inhibition (Fig. the PARP to SMARCA5 beyond while more response was expansion spreading Surprisingly, in GFP-RNF168 impaired that RNF8 cells. much found we affect control expansion, chromatin not in was did MDC1 to compared PARPi of tracks reduced spreading albeit S5C), the same the MDC1 Fig. endogenous material in of (supplementary the expansion spreading the little while along PARPi, showed of laser-tracks SMARCA5-GFP efficient that presence found Consistent for we by SMARCA5, chromatin. required on marked damaged effect is selective when into PARP1 a or with SMARCA5 inhibits that 2011), of material indicates was that (supplementary al., spreading This depleted width et concentration S6). was track PARP2, (Boehler Fig. PARPi not PARP2 on but low than PARP1, impact rather a similar a supplementary PARP1 A using to 3B–D; SMARCA5-GFP (Fig. S5B). 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DNA this implicate of in sites findings at chains expansion These poly(ADP-ribose) chromatin of 3B). (Fig. synthesis a damage-induced the process highlighting for depletion, DNA ATP role to the novel similar suppressed chromatin of PARPi expansion Remarkably, ATP 3A,B). with of (Fig. cells depletion treatment fixed a by chemically inhibited in is abolished was expansion and it observed as minutes The phenomenon 3 biological 3A,B). first (Fig. the irradiation within following expanded rapidly tracks PAGFP-H2A ae oehr u nlsssget htPRsynthesis PAR that suggests analysis our together, Taken ora fCl cec 2 (4) 126 Science Cell of Journal c 2X(Huen H2AX cuac fSAC5adfcoso h N18signaling RNF168 the of chromatin. damaged factors throughout and cascade SMARCA5 of the and occupancy chromatin of expansion damage-induced DNA the Thus, that regulate mechanisms 3E,F). 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(Fig. RNF168 endogenous and between SMARCA5 interaction it IR-induced with cells, the non-irradiated treatment abrogated in nearly although interaction the that, enhanced slightly found PARPi and we SMARCA5 GFP-tagged Remarkably, between whether interaction RNF168. investigated the we that affects chromatin, inhibition PARP damaged SMARCA5 experiment into 4C,D). of RNF168 (Fig. spreading the and affects RNF168 reciprocal selectively and PARPi endogenous that Given SMARCA5 with the streptavidin- associates endogenous SMARCA5 that in with SMARCA5 demonstrated interacts showed and we RNF168 RNF168 tagged Furthermore, IR-dependent endogenous 4B). the (Fig. confirmed between analysis Immunoprecipitation blot 4A). interaction (Fig. western an by light of by UV-C to revealed exposure followed interactome not after but approach IR, SMARCA5 RNF168 to and cells the This RNF168 the between spectrometry. considered interaction screened test mass we To we RNF168. quantitative events, with hypothesis, interact chromatin these may this both SMARCA5 cascade SMARCA5 into that gain of possibility To signaling between insight DSBs. distribution of RNF168 link mechanistic signaling RNF168-dependent the the functional and remodeling of regulates a factors PARP1 suggests and that SMARCA5 fact and The damage- DNA a manner in PARP-dependent interact RNF168 and SMARCA5 N18fo nrae,I-rae rMN-rae cells or subsequently MMNG-treated or We SMARCA5 treatments IR-treated enzymes. these untreated, PARP1, after PARP from that shortly of GFP-tagged RNF168 indicating increase activation 4F), immunoprecipitated (Fig. after moderate in proteins IR a result we PARylated to and (WCE), in exposure extracts treatment, increase cell whole these significant MNNG In a agent of PARP1. and alkylating of observed SMARCA5 either DNA that of the status to of or PARylation RNF168 the IR PARylation compared to and cells MNNG involve exposed We may SMARCA5 proteins. 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RNF168 of identify modified substrate we not damage-induced Thus, is induction. suggesting protein damage SMARCA5, RNF168 this of PARylated that detect PARylation to robust in failed damage-induced we increase more Conversely, DNA cells. IR, reproducible PAR the non-irradiated to following a compared reflecting of Nonetheless, detected MNNG. likely induction after we PARP1 most the of WCE, activation mirrored after in RNF168 closely of synthesis PARylation treatment stronger The MNNG 4F). (Fig. treatment nvivo in ora fCl cec 2 (4) 126 Science Cell of Journal neato ewe MRA n A olwn DNA following PAR and SMARCA5 between interaction ´ ta. 08.T netgt the investigate To 2008). al., et iemhrytge sfrsof isoforms mCherry-tagged tive ciePR PR-u) which (PARG-mut), PARG active dniidSAC5a putative a as SMARCA5 identified igt hs tutrs(Ismail structures these to ding eut nrdcdsteady-state reduced in results rtistruhu aae hoai n hi physical, their damage. DNA and to response chromatin in interaction damaged PARP-dependent these of The throughout expansion reduced PARP-dependent mutually the proteins 6E,F). is with (Fig. knockdown line accumulation in RNF168 is DSB-tracks dependent and RNF168 SMARCA5 in that that finding 6C,D). assembly (Fig. found depletion SMARCA5-GFP we SMARCA5 was by Conversely, laser-tracks impaired in Corroborating severely markedly accumulation 6A,B). was also (Fig. GFP-RNF168 IRIF findings, depletion into these SMARCA5 RNF168 that by response. of suggests DSB impaired the accumulation of This contrast, stages early S8A). In the affect Fig. not does material SMARCA5 (supplementary IR-induced cells normal western showed agreement, In analysis 5A,B). blot (Fig. IRIF into accumulation RNF8 i.4) hs idnsprovide findings These 4G). Fig. fsvrlfcosivle nteRF6 acd.W on that found neither efficient We affected cascade. knockdown RNF168 we an SMARCA5 the formation in end, IRIF involved on factors this for depletion several of SMARCA5 To of required damage. impact the the DNA is examined raises to RNF168 response SMARCA5 RNF168-ubiquitin and whether SMARCA5 question between interaction The mutually is DSBs dependent at accumulation RNF168 and SMARCA5 2XaetepiaykontresfrIR-induced for targets known primary and the H2A that are Given 7A,B). (Fig. reduced H2AX substantially cells also SMARCA5-depleted was found IRIF in then in we conjugation recruitment, ubiquitin RNF168 that impaired with agreement In DSBs at accumulation stimulates SMARCA5 Ayain cnroeegsi hc MRA id to binds of damage. DNA SMARCA5 of substrate sites which at damage-dependent RNF168 in on DNA present emerges chains novel PAR scenario a a Given structures. is PARylation, these binds RNF168 directly it that where to damage, recruitment DNA its for of chains sites PAR of synthesis the requires SMARCA5 LGRF.Mr hn10nce eesoe per experiments. scored independent were in nuclei treatment 150 than More foci FLAG-RNF8. 10 than more with c foci/nucleus average of The A. number in formation IRIF (right) FLAG-RNF8 10 of bar: representation Scale (Mailand 2007). IRIF al., for visualize et immunostained to later FLAG-RNF8 h or 1 MDC1 and to IR exposed Gy siRNAs, 1 indicated were the RNF8 with against transfected shRNA an expressing and cells FLAG-RNF8 U2OS doxycycline-treated DSBs. or at cells accumulation RNF8 promote or not MDC1 does SMARCA5 5. Fig. 2XadMC,weestepretg fcells of percentage the whereas MDC1, and H2AX c 2Xuiutlto n BRCA1 and ubiquitylation H2AX nvivo in c 2X(et,MC mdl)or (middle) MDC1 (left), H2AX 6 6 c vdnesgetn that suggesting evidence c ...i rsne for presented is s.e.m. for presented is s.e.m. 2XadMC,nor MDC1, and H2AX m 2Xlvl nthese in levels H2AX .( m. B Quantitative ) c H2AX, c ( H2AX, U2OS A) Journal of Cell Science el splmnaymtra i.S) sepce,dpeinof same depletion the expected, As in S7). impaired Fig. material significantly (supplementary were cells assembly conjugation ubiquitin BRCA1 formation though and IRIF even 53BP1 cells, in Surprisingly defect SMARCA5-depleted 2011). any its in observe al., not et did (Nakamura we SMARCA5- for however, 7E,F) in found impaired (Fig. we 2009). was cells ubiquitin conjugation, IRIF al., depleted into ubiquitin et recruitment conjugated in Wu BRCA1 defects 2007; that the al., to et with (Sobhian Consistent binds DSBs that at ubiquitin accumulation which notion RNF168-dependent complex, the the with shown). in not consistent (data cascade As operates proteins, 2007). SMARCA5 individual al., reduce the not et did or co-depletion (Huen RNF8 that c RNF168 and after 7C,D) found SMARCA5 (Fig. observed expected, we that knockdown data, to RNF168 blot similar IRIF di-ubiquitylation, western IR-induced our and by decreased with knockdown status agreement SMARCA5 ubiquitylation In its analysis. examined and cells 07 twr ta. 09 age l,20) we 2007), al., et Wang 2009; al., et al., et immunoprecipitated Mailand Stewart 2007; al., et Huen 2007; 2009; al., et (Doil ubiquitylation 2Xuiutlto eodta bevdatrdpeinof depletion after observed that beyond ubiquitylation H2AX enx nlzdrcuteto h RAP80–BRCA1 the of recruitment analyzed next We c 2Xfo nrae n IR-exposed and untreated from H2AX c 2Xmono- H2AX MRA/N2 euae N aaesgaig897 signaling damage DNA regulates SMARCA5/SNF2H n euae h w ao ahaso S ear Rand HR repair, DSB IR of against pathways major cells two protects the SMARCA5 regulates that and revealed study Our Discussion DSBs. at accumulation BRCA1 for to essential activity is ATPase failed SMARCA5 the that of suggesting SMARCA5 8A–C), (Fig. ATPase-dead defect BRCA1 this 8A–C). rescue of (Fig. SMARCA5 expression impaired wild-type rescued fully Conversely, of was expression severely defect exogenous This by 8A–C). (Fig. depletion IRIF into shown recruitment As SMARCA5 2010). al., et before, (Lan SMARCA5 endogenous GFP-tagged of expressed depleted cells is in We SMARCA5-K211R ATPase-dead activity, or accumulation. SMARCA5 wild-type remodeling BRCA1 chromatin for its required is for essential response SMARCA5, SMARCA5-RNF168 the when to sites impaired. accumulation required 53BP1 damage that levels promote or at that differ, ubiquitin exist may mechanisms recruitment for 2007; 53BP1 alternative al., or threshold BRCA1 et either This the promote Kolas S7A,B). Fig. that 2007; material al., (supplementary suggests 2007) et 53BP1 al., (Huen et also Mailand but IRIF conjugation, in ubiquitin accumulation impaired only not RNF8 ial,w sesdwehrteAPs ciiyof activity ATPase the whether assessed we Finally, nlzd cl as 5 bars: Scale analyzed. against siRNAs with ( transfected RNF168. and used were GFP experiments. independent from cells ( individual 10 means least are at Values units time. against fluorescence plotted Relative are C. (RFU) in results of representation foci/ of number average The A. nucleus in formation IRIF RNF168 rti sebya h aae ra ( area. damaged the of at recording assembly real-time protein to siRNAs, subjected indicated and the laser-irradiated with transfected were ( GFP-RNF168 (control). * siLuc experiments. with independent compared in treatment per scored xoe o1G Rad1hltrimnsandfor immunostained later h ( IRIF. 1 visualize and to IR RNF168 Gy 1 to exposed DSBs. at assembly RNF168 ( promotes SMARCA5 6. Fig. E 2Sclswr rnfce ihteidctdsiRNAs, indicated the with transfected were cells U2OS A) si ,ecp htUO el xrsigSMARCA5- expressing cells U2OS that except C, in As ) 6 F ...i rsne.Mr hn10nce were nuclei 150 than More presented. is s.e.m. si ,ecp htclsfo were E from cells that except D, in As ) m m. B uniaierpeetto of representation Quantitative ) C 2Sclsexpressing cells U2OS ) Quantitative D) 6 P ...of s.e.m. , 0.05, Journal of Cell Science a sda odn oto.( control. loading c a as used was with IP to subjected were ( (control). siLuc foci/nucleus with of number average The A. in needn xeiet sson ( shown. is experiments independent 10 bar: Scale IRIF. visualize to (FK2) ubiquitin conjugated for immunostained later h 1 and IR Gy 1 898 nue Ayain hoai eoeigadteRNF8/ damage- the and DNA 8D). (Fig. remodeling between cascade RNF168-signaling chromatin link a which unveil PARylation, functional findings assembly, induced and Our PARP- physical activity. BRCA1 histone novel ATPase a subsequent RNF168-dependent SMARCA5 in requires regulate and compartment to ubiquitylation manner chromatin an RNF168 dependent in DSB-flanking uncover the interact al., RNF168 we regulating and the et SMARCA5 addition, in DSBs. (Lan at SMARCA5 In cascade reports signaling of 2011). recent role two al., unrecognized with et line Nakamura in 2010; is which NHEJ, promotes SMARCA5 7. Fig. 2Xadete oouiutltdo di-ubiquitylated or mono-ubiquitylated either and H2AX ora fCl cec 2 (4) 126 Science Cell of Journal el rnfce ihteidctdsRA eeepsdt 0G R ae hoai-nihdetat CE eepeae.CEE prepared. were (CEE) extracts chromatin-enriched later h 1 IR. Gy 10 to exposed were siRNAs indicated the with transfected Cells C) c 2Xatbd.Wsenbo nlsso Pwith IP of analysis blot Western antibody. H2AX c rpia ersnaino relative of representation Graphical D) 2Xuiutlto n RA cuuaina DSBs. at accumulation BRCA1 and ubiquitylation H2AX E si ,ecp htclswr muotie o RA.( BRCA1. for immunostained were cells that except A, in As ) 6 ...i rsne.Mr hn10nce eesoe e ramn nidpneteprmns * experiments. independent in treatment per scored were nuclei 150 than More presented. is s.e.m. c 2Xwr omlzdt hto oto el,wihwsstt .Temeans The 1. to set was which cells, control of that to normalized were H2AX c 2Xuiutlto eesdtrie sn eut rmC aiso unmodified of Ratios C. from results using determined levels ubiquitylation H2AX c 2Xatbd,adCEwt MRA nioyaeson oca staining S Ponceau shown. are antibody SMARCA5 with CEE and antibody, H2AX 3P,BC1 RA,RD1adp3,ete eoeo after or affect before either not p53), RNF168, did and RNF8, RAD51 (MDC1, SMARCA5 BRCA2, factors BRCA1, of DDR 53BP1, loss key of that This levels found to steady-state compromised. response we in be expression However, gene may IR. orthologues, altered from response stem human DSB indirectly IR, could the to its of hypersensitivity that effects of displayed genotoxic indicating cells the one against SMARCA5-depleted cells IR. that 2006). protects al., also evidence et SMARCA5, Haaften (van provide IR against We worms protects that factor S- a dniidtruhagntcsre in screen genetic a through identified was damage ISW-1 DNA against protects SMARCA5 ( A el eetasetdwt h niae iNs xoe to exposed siRNAs, indicated the with transfected were Cells ) m F .( m. si ,ecp htclsfo eeanalyzed. were E from cells that except B, in As ) B uniaierpeetto fF2II formation IRIF FK2 of representation Quantitative ) P 6 .elegans C. , ...o two of s.e.m. .5 compared 0.05, sa as Journal of Cell Science nue yteI-S DSBs the chromosomal by site-specific both induced DSBs to to recruited SMARCA5 is of SMARCA5 recruitment the and PARP al., et DSBs (Lan to NHEJ 2011). and recruited al., HR et both is Nakamura by SMARCA5 repair 2010; DSB S8), that regulates we it shown Fig. Consistently, where have repair. by DSB material others IR promote against to and (supplementary DSBs cells at human IR directly protects acting to SMARCA5 that cells suggesting of exposure ( control. loading a as used 5 was bar: Tubulin Scale A. and in DAPI. recruitment ( PARP1 cells and scored. triggers were of BRCA1 experiments extracts for independent cell two stained whole least and at using IR from Gy cells 100 2 than to More exposed plasmids, DSBs. SMARCA5-K211R-GFP at accumulation or ( BRCA1 SMARCA5-WT-GFP for GFP, required is either activity and remodeling chromatin SMARCA5 8. Fig. rmtsefcetrpi fDBb HJadH setx o details). for also SMARCA5 text interaction signaling. (see RNF168 DSB HR and RNF168-dependent and SMARCA5 regulating NHEJ stimulates signaling thereby and by RNF168 simplicity), RNF168, DSB the for of of of shown PARylation repair factors are through efficient and of BRCA1 likely promotes SMARCA5 accumulation and most of RNF168 the chromatin, distribution only promoting DSB-flanking spatial BRCA1; ubiquitylation, throughout the RAP80, RNF8-induced promotes RAD18, amplifies UBC13, PARP1 which (RNF168, complex. recruitment, cascade RAP80–BRCA1 RNF168 the to including leads factors, This ubiquitin-binding histones. H2A-type ubiquitylates which B uniaierpeetto fBC1II omto nA h ecnaeo el ihmr hn1 oiprncesi rsne.Errbr niaetes.d. the indicate bars Error presented. is nucleus per foci 10 than more with cells of percentage The A. in formation IRIF BRCA1 of representation Quantitative ) ce IorA c si 2Xfrain AP lct oa hoai xaso,whereas expansion, chromatin local elicits PARP1 formation. H2AX Incessadt laser-induced to and nucleases SI MRA/N2 euae N aaesgaig899 signaling damage DNA regulates SMARCA5/SNF2H MRA n MRA-F eeswr oioe ywsenbo analysis blot western by monitored were levels SMARCA5-GFP and SMARCA5 C) oe o h oeo MRA nteDBrsos.ADBi chromatin in DSB A response. DSB the in SMARCA5 of role the for Model D) htSAC5i ail erie oDB namne that manner a in demonstrating DSBs by to findings recruited these rapidly extend is DSBs We SMARCA5 at 2010). that assembly in in not al., SMARCA5 was ACF1 et abrogates al., ligase it (Lan for loss formally et ACF1 ubiquitin role although that a DSBs, (Nakamura shown E3 to suggested 2010; breaks recruitment report RNF20 SMARCA5 al., another DNA the et whereas, to 2011), Nakada for recruitment 2010; role SMARCA5 al., a et S3C,D) implicated Lan Fig. 2010; material Sa al., supplementary et 1F; (Fig. (Erdel damage DNA ´ ce-oiae l,21) aauaadco-workers and Nakamura 2011). al., et nchez-Molina ( 2Sclswr rnfce ihteidctdsiRNAs indicated the with transfected were cells U2OS A) c 2Xpooe h cuuaino D1adRNF8, and MDC1 of accumulation the promotes H2AX m m Journal of Cell Science 900 tDAdmg sites. damage accumulation SMARCA5 DNA to at contributing RNF168 damage, DNA as of such sites proteins at PARylated with PAR-binding associate the a and contain of sequence may SMARCA5 one of partners Alternatively, within binding compartment. spreading many and chromatin RNF168, damaged as is the such proteins association it the PARylated for responsible with that that be may suggests SMARCA5 indicated This in regions S9). other sequence Fig. in material binding SMARCA5 (supplementary of DSB-tracks accumulation PAR PARP-dependent for putative mutation dispensable this, with the line In of DSBs. at accumulation SMARCA5 for n osdrn htSAC5itrcswt N18in RNF168 tempting with is it manner, interacts PARP-dependent a SMARCA5 in chromatin that PARP1-dependent damaged partially that considering is Given MDC1. DSBs and the and to RNF8 for the recruitment as dispensable such SMARCA5 is affects factors factors, it other RNF168-associated PARP while of of spreading regions, that subset chromatin damaged specific suggests supplementary into a This 3; factors, of (Fig. downstream S5B,C). distribution its regions Fig. of chromatin several material and DSB-flanking RNF168 found of we into that addition, as as distribution, In SMARCA5 well impair 3). selectively (Fig. to inhibition damage PARP DNA as sites process laser-induced at this expansion in of chromatin involved reduces is moderately PARP inhibition that PARP showed We 2006). al., or et ATM of independently expansion breaks. chromatin suggests DNA to ubiquitylation at work accumulation RNF168-dependent BRCA1 our contributes elicits the DSBs, efficient which PARP1 modulates response, at regulates PARP1 that assembly DSBs that SMARCA5 BRCA1 Given to and and SMARCA5 2008). RNF168 of recruitment recruitment al., rapid SMARCA5 line et the in is in breaks (Haince strand role DNA its to PARP1 with of recruitment rapid The cascade signaling RNF168 DSBs the at to SMARCA5 links PARP report recent binding a PAR for It vitro dispensable but in binding. was sequence, sequence PAR this PAR-binding that for demonstrated putative domain finger a canonical zinc harbors a PBZ contain not PARylated or does SMARCA5 with macro-domain clear. yet interacts not SMARCA5 is RNF168 how precisely and manner. 2010). SMARCA5 PARP-dependent However, and that damage- DNA found al., a we in interact this, RNF168 et with for Consistent those potentially Polo substrate RNF168. chains, on PAR novel to 2009; binds SMARCA5 a that al., suggesting as obtained and et PARylation, RNF168 damage-induced DNA identified Gottschalk we 2010; Interestingly, al., Chou 2009; al., et et their (Ahel sites for damage activity DNA CHD4, to PARP recruitment efficient and require Hottiger, histone and ALC1 and regions enzymes, and PAR-binding (Messner for remodeling contain response non-histone itself chromatin DSB targets Two other the 2011). only in several not involved PARP1 also proteins that but between known is PARylation, crosstalk It sites? the DSB SMARCA5 how sites. orchestrates it DSB unravel at activities 2010), activity to al., ACF1 and/or et accumulation and interest Nakada RNF20 at 2010; of PARP1, assembly al., be et SMARCA5 and (Lan will to RNF20 breaks that contribute strand Given DNA also 2). (Fig. ACF1 PARP1 potentially of activity the requires rvoswr ugse htlsrmcoirdainevokes micro-irradiation laser that suggested work Previous at accumulation SMARCA5 affect activity PARP1 does How (Gagne ora fCl cec 2 (4) 126 Science Cell of Journal ´ ta. 08,sgetn hti a o erequired be not may it that suggesting 2008), al., et c 2X(Kruhlak H2AX nvivo in evidence htacneuiesre fdsic hoai configurations chromatin distinct of series ubiquitin consecutive suggest the findings a These in different DSBs. that recruit involved to enzymes accumulation, all remodeling BRCA1 chromatin are promotes which that response RNF168, MDC1, Thus, manner. and dependent mutually that RNF8 a are in showing RNF168 DSBs to and by recruited SMARCA5 DNA findings and that PARP- and these a manner in extend damage-dependent RNF168 a with We interacts in directly 2010). tracks SMARCA5 2012a; al., DSB-containing al., et et to (Luijsterburg Polo recruited manner to RNF8-dependent is recruitment and and PARP- directly its RNF8 CHD4 and Similarly, to 2010). MDC1 al., binds et with (Xu complex MDC1 requires a shown DSBs was forms it a p400 Mechanistically, play DSBs. that factors at assembly remodeling BRCA1 chromatin in role distinct three Thus, 2010). eedn neato ewe hs atr sstrictly of is binding involves factors likely damage- 4). 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RNF168, DSBs, this, of at accumulation BRCA1 of and the support affects their In loss for DSBs. be important SMARCA5 of is may at factors It spreading retention these response. of sustained the RNF168 distribution for the correct the of required that factors is signaling and SMARCA5 repair that speculate to RA sebya Ss(oo n Co and (Doyon NuA4 DSBs the been at and has assembly of ubiquitylation that 2010). BRCA1 RNF8/RNF168-dependent subunit factor promote al., remodeling catalytic to et chromatin shown the Smeenk another 2012a; is is al., complex, which the et p400, promoting Luijsterburg Likewise, 2010; by (Larsen RNF8/RNF168 al., of accumulation level et the BRCA1 at cascade signaling shown affects ubiquitin recently have CHD4 we here, that reported SMARCA5 to addition In the in cascade involved RNF168 remodelers chromatin other and SMARCA5 upiigy hl APihbto boae h IR-induced the abrogated inhibition PARP while Surprisingly, ˆ te ´ 04 ue al., et Xu 2004; , c H2AX/ Journal of Cell Science rz eeue tacnetaino 10 of concentration (Santa a dehydrate salt at ammonium used ADP-HPD were and Cruz) Biosciences) EMD (NU7026, eeae ihaMr oeokdT:apielsr(l laser Ti:Sapphire modelocked Mira a length with generated euae h ellrrsos oDB n ssc promotes such as interplay and insult. genetic DSBs the to to response in cascade response that stability ubiquitin genomic cellular RNF8/RNF168 the reveal the regulates DSBs. and findings of remodeling SMARCA5-mediated chromatin repair PARylation, of our damage-induced and DNA functions between signaling together, the PARP-independent to Taken contribute and how remodel precisely SMARCA5 address PARP-dependent SMARCA5 should assembly RNF8/RNF168 studies the and the Future promote BRCA1 to p400 cascade. and DSBs to signaling of promote CHD4, vicinity RNF8 remains the it in to how MDC1, chromatin However, established sites 2012). when Attikum, be these DSBs van and at at (Luijsterburg established act be RNF168 to need are u naLiaS5cnoa irsoeeupe iha environmental an with equipped 37 microscope confocal to SP5 set Leica chamber 4 a (AMG, on the microscope out fluorescence carried using fl EVOS Scientific) an with Westover taken were and pictures NaCl Brightfield micro-irradiation 0.9% laser scored. with and were washed Microscopy cells were 20 than cells more later of days Colonies Blue. 7 and Methylene density PARPi. low with or at stained seeded IR trypsinized, h, to 48 exposed for transfected were cells KV, VH10-SV40 200 assay International, survival (YXlon Cell generator X-ray Gy/min). 1.1 YXlon rate a dose mA, by 4 delivered was IR DSBs of Generation ATM stated. otherwise 10 O’Connor of unless Mark concentration from a transfection gifts at were used siRNA Cells (KU-0058948) and 2009). PARPi after al., and et h (KU-55933) (Doil inhibitor 48 described as examined used were were RNF168 against 5 siRNAs 2007), MWG). al., et (Sartori (XRCC4) 59 used: were sequences the siRNA UUU-3 to following according respectively, The CGUACGCGGAAUACUUCGA-3 Transfection), instructions. (Polyplus (Open manufacturer’s 2000 JetPEI using Lipofectamine SMARCA5 performed and (Invitrogen), were human (Invitrogen) transfections RNAiMAX plasmid Lipofectamine for stably and (Qiagen), siRNA was cDNA HiPerfect was cells. SMARCA5-GFP U2OS The (Clontech). pPAGFP-H2A in pEGFP-N1 2012b). expressed into pGFP-RNF8. cloned al., pGFP- in was vector et Biosystems) described mCherry previously the (Luijsterburg in with previously PAGFP H2A with were was GFP GFP replacing pmCherry-RNF168 by pGFP-UBC13 replacing and Valerie generated pmCherry-RNF8 Yasui. Tateishi, by 2009). Satoshi Akira al., made Jetten, et and Anton (Marteijn Hendzel Lukas, described Jiri expression Michael from Schreiber, pSMARCA5-K211R-GFP obtained pGFP-RNF8, were XPA Lukas. and vectors Jiri in FLAG- pmCherry- pSMARCA5-WT-GFP from GFP-MDC1, deficient pmCherry-PARG-WT, gifts PARG-mut, expressing pGFP-PARP1, are were stably pGFP-RAD18, GFP-53BP1 which cells pGFP-RAP80, or cells, U2OS BV). GFP-RNF168 2003). (Bodinco XP2OS-SV RNF8, al., FCS in 10% et containing expressed (Rademakers (Gibco) was DMEM in GFP-XPA grown immortalized were XP2OS-SV and fibroblasts VH10-hTERT VH10-SV40, HeLa, HEK293, U2OS, chemicals and culture Cell Methods and Materials irsgaiie(NG im)wsue tacnetaino 100 of concentration a at used was Sigma) (MNNG; nitrosoguanidine a e o0 ooti eaiefursec nt RU.Takwdhwas width damage. 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Thanos 53BP1, PARP1 Durocher, Santa RNF168, Life and Daniel to and (Abcam) from Enzo antibodies MDC1 (FK2, (Calbiochem The and H3 Signaling). PARP2 BRCA1 histone and SMARCA5/SNF2h, (Trevigen), (Novus), ubiquitin PAR (Roche), Sciences), GFP BioTAGnology), (IBA w asatrtefrttaseto el eetasetdwt pGFP, with transfected were day microscopy. fluorescence cells 1 and and immunoblotting transfection by DNA analyzed first and plasmid (SMARCA5-1). irradiated the later pSMARCA5-K211R-GFP SMARCA5 against or after siRNA pSMARCA5-WT-GFP days using twice Two transfected were Cells assay timepoint Complementation each at previously established described was as significance ANOVA. Statistical software one-way 2010). using Stacks al., home-made et using (Smeenk analyzed were IRIF analysis IRIF to antibodies using performed were c analysis blot western and Immunofluorescence Antibodies NaCl, CaCl mM mM (137 medium 1.8 depletion KCl, ATP mM in incubated 5.4 and PBS with rinsed were Cells depletion ATP a¨ l eue no etn aoh aesi aei Schreiber, Valerie Tateishi, Satoshi Halozanetis, Jasin, Jetten, Thanos Maria Anton Kanaar, Durocher, Roland Legube, Dan Gae¨lle Modesti, Lukas, Mauro Jiri Stark, O’Connor, Jeremy Mark thank We Acknowledgements or DR-GFP the I-S of of repair copy the integrated measure stably to used a were either reporter containing EJ5-GFP lines assays cell end-joining HEK293 non-homologous and recombination Homologous fluorescence the analyze and to membranes the Li-Cor scan for signals. to with used processed contained equipped subjected was and buffers software Biosciences) 3.0 SDS-PAGE (Li-Cor All Odyssey imager by Tween. were Odyssey resolved 0.1% The were M with immunoblotting. proteins 1 lysates supplemented Bound with TBS supplemented were inhibitor. buffer and beads PARG RIPA Cleared The times) times), h. (2 (three 2 buffer NaCl cocktails. RIPA for with (Chromotek) supplemented washed 8.0, beads then pH SDS) inhibitor Trap Tris, 0.1% GFP mM deoxycholate, with minutes. (10 immunoprecipitation 0.1% protease 5 buffer X-100, RIPA for Triton in MNNG with lysed 1% with then NaCl, treated and mM or PBS 450 irradiated pGFP- in either collected and days were pSMARCA5-GFP Cells 2 pGFP-PARP1, after pGFP, and with RNF168 transfected were Cells assay PARylation (Thermo MaxQuant spectrometer with performed mass glycine was analysis Velos M Data 0.1 amino RNF168 LTQ-Orbitrap 2003). with al., software. an by beads et from on (Ong interaction, labeling eluted Scientific) analyzed were Fisher isotope mass days) and RNF168-SMARCA5 stable 7 For 2.5) (SILAC; (pH to culture immunoblotting. the cell subjected in for acids cells of processed proteins were from Bound Strep-Tactin and Beads buffer. immunoprecipitates analysis sample h. SDS-PAGE (Chromotek), in 1.5 boiled by spectrometric for and beads resolved buffer antibody EBC were Trap RNF168 with times or four GFP supplemented washed BioTAGnology) EDTA) subjected with (IBA buffer mM were 1 lysates Sepharose EBC Cleared NP-40, immunoprecipitation in cocktails. 0.5% inhibitor NaCl, lysed to phosphatase mM were and 150 protease 7.5, cells with pH interaction Tris, RNF168-SMARCA5 mM (50 the study To studies interaction Protein of immunoprecipitation for used and c prepared were extracts Chromatin-enriched assay Ubiquitylation otemcocp hme n ujce omlihtnmicro-irradiation. multiphoton to subjected and chamber microscope the to 0m EE n 0 C)o okmdu 17m al . MKCl, mM 5.4 NaCl, mM (137 medium mock or FCS) CaCl 10% mM 1.8 and HEPES mM 20 2X hsh-itn 3S0 (Millipore), S10p H3 phospho-histone H2AX, 2Xa ecie rvosy(meke l,2010). al., et (Smeenk previously described as H2AX ˚ sdsrbdpeiul Lisebr ta. 02) el eete transferred then were Cells 2012b). al., et (Luijsterburg previously described as C 2 . MMgSO mM 0.8 , 2 . MMgSO mM 0.8 , 4 ,2 0m MD - g l u c o s e ,2 0m MH E P E Sa n d1 0 %F C S ) 4 0m exguoe 0m NaAz, mM 30 deoxyglucose, mM 60 , a Tbln(im) Streptavidin (Sigma), -Tubulin ce -nue Ssb HR by DSBs I-induced ce xrsinvector expression I Journal of Cell Science oo,Y n Co and Y. Doyon, 902 re,F,Shbr,T,Mrh . ans,G n ip,K. Rippe, and G. La¨ngst, C., Marth, T., Schubert, F., Erdel, R., Pepperkok, H., D. Larsen, P., Menard, S., Bekker-Jensen, N., Mailand, C., Doil, Varga- and G. Garcı´a-Jime´nez, Dellaire, I., C., Kukimoto, A., R. Poot, N., Collins, E., K. Hurov, C., A. Nottke, X., Tan, E., N. Dephoure, B., Adamson, M., D. Chou, Bresson, M., J. Saliou, S., D. Biard, O., Mortusewicz, R., L. Gauthier, C., Boehler, and J. D. Picketts, W., N. D. Mailand, and Speicher, S. S., Bekker-Jensen, W. Lane, A., M. Lazzaro, O., Barak, X. Shen, and Y. Bao, Flynn, I., Ahel, E., Garcia-Wilson, E., S. Polo, N., Wiechens, Z., Horejsı´, D., Ahel, References [grant at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.109413/-/DC1 online available Research material Supplementary Scientific M.S.L.]. and for J.A.M. from to grants 863.11.007 Organization Netherlands 916.96.120, Veni Program numbers and The Netherlands Science H.V.A.]; Frontier from to Human The to CDA00048/2009 grant The 864.07.001 number from Vidi grant [grant number a CDA [grant a Research by H.V.A.]; Scientific funded for was Organization work This Funding wrote H.V.A. and M.S.L. G.S., data. manuscript. the the N.M., analysed R.J.R. T.C., H.V.A. M.S.L., and and J.A.M., T.C. W.V. W.W.W., N.S., G.S., M.S.L., experiments. 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