Ato o orsodne([email protected]) correspondence for *Author UK. 5EH, DD1 Germany. Dundee Wuerzburg, Dundee, of University Centre, Black James Sir hitpeLachaud Christophe UBZ anemia ubiquitin-binding Fanconi SLX4 in two mutated the domains for roles functional Distinct REPORT SHORT ß eevd1 oebr21;Acpe pi 2014 April 5 Accepted 2013; November 12 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This 2 Fanconi cause FANCP) 1 as known SLX4, (also 2013). SLX4 bi-allelic al., of consequently, in repair and, et mutations the (ICLs) junction for Wyatt crosslinks Holliday important inter-strand is 2013; for DNA nucleases, al., required associated with are et together that (Castor made nucleases, incisions, resolution dual two the the coordinates scaffold by and SLX4 MUS81 the of tethering to The during SLX1 BLM–TOP3–RMI1– forks. arise replication to the collapsed thought of are and repair by Holliday junctions these resolve dissolution SLX1 complex; (BTR) to RMI2 escape intermediates. together that repair act junctions example, DNA for branched MUS81–EME1, cleave can al., Mun et (Fekairi 2009; SLX1 structure-selective and the MUS81–EME1 coordinates XPF–ERCC1, that nucleases scaffold a is SLX4 Human INTRODUCTION ICL UBZ, anemia, Fanconi Ubiquitin, FANCP, SLX4, E3 WORDS: KEY and repair. ICL ligands the for to crucial ubiquitylated point are unidentified they that junctions ligases and Holliday currently recruitment, of of SLX4 resolution on existence light the shed for data required These is UBZ-2 SLX4 it The ubiquitin but fibroblasts. to sites bind murine chains. not ICL in does to repair K63-linked domain recruitment ICL for efficient SLX4 for for preference and required a is binds UBZ-1 with SLX4 Furthermore, of polymers, show domain of UBZ We ubiquitin second ubiquitylation BRCA1. the to not and the but RAD18 (UBZ-1) RNF8, first either the ligases that require not. E3 is the not patients or FA FANCD2 does from but cells induction recruitment in ICL SLX4 of SLX4 of sites form to UBZ-deleted recruited the deletion is Here, that SLX4 damage. human DNA SLX4 of that that sites show an to we domains recruitment protein ubiquitin-binding have in implicated UBZ4-type patients are tandem in FA two result Some removing nucleases, DNA repair (ICLs). inter-strand of DNA repair crosslinks defective for the to due scaffold (FA), anemia a Fanconi SLX4, in Defects ABSTRACT elvSchindler Detlev eateto ua eeis nvriyo urbr,Biozentrum, Wuerzburg, of University Genetics, Sciences, Human Life of of Department College Unit, Ubiquitylation and Phosphorylation Protein MRC 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,21–87doi:10.1242/jcs.146167 2811–2817 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. o ta. 09 vnsne l,20) hc together which 2009), al., et Svendsen 2009; al., et ˜oz 2 n onRouse John and 1 ensCastor Dennis , nvitro in rcnrbt oILrepair, ICL to contribute or 1, * 1 aoiaHain Karolina , nvivo in . 1 vnMun Ivan , rtissc sFN ostso N aae(Hofmann, mitomycin-C- damage in DNA foci of sub-nuclear sites to to recruitment FAN1 SLX4 2009). as such for vital proteins are domains repair. UBZ tandem ICL the two in of of that function both first SLX4 or SLX4 al., the one et of in Therefore, (Stoepker most 1A) 2011). deletion (Fig. and domains small second ubiquitin-binding the UBZ4-type a of FA by all D’Andrea, with removes caused siblings and German 457/1–3) rare three (Moldovan (patients a described predisposition previously 2011), We al., cancer 2009). marrow et bone and Stoepker defects, failure 2011; developmental by al., characterized et condition (Kim (FA) anemia xlisteILrpi eet nteecls nti td,we study, this In cells. these defective in issues. SLX4 defects these whether UBZ-deleted address junction repair expressing clear ICL Holliday cells the yet FA explains for in not in SLX4 and/or domains is of recruitment repair UBZ it the DNA Furthermore, of of sites resolution. for to both protein damage, the or of recruitment DNA one for required whether are SLX4 known human one. not only have is eukaryotes other It some in homologs SLX4 whereas of absence 2013). the al., in et even it (Wilson because telomeres) damage SLX4 of to DNA sites for (corresponding the difficult foci at is forms foci visualization sub-nuclear this to of damage, applies formation also DNA can the this FANCD2 by whether as visualized such clear proteins be yet of not recruitment the is domain Although 2001; UBZ4 it SLX4. a but al., through cells damage human et DNA in of (Garcia-Higuera sites to the ICLs nuclease recruits FANCD2 FAN1 Mono-ubiquitylated to 2002). complex al., response core et Taniguchi FA in the the Lys561 by of component mono-ubiquitylated at central is a that is FANCD2 pathway latter FA of The UBZ 2011). mono-ubiquitylation single al., and et the (Yamamoto SLX4 both require chicken to in reported domain was cells DT-40 treated omdasrp ln h ae rc,wihas otie the contained also which track, in variant laser shown the histone As along phosphorylated panels), laser. (middle stripe GFP UV-A a not 355-nm but formed panels), a (upper using GFP–SLX4 1B, nuclei Fig. cell were along al., in micro-irradiated GFP–SLX4 track and et a expressing (TMP) (Thazhathveetil trimethyl-psoralen stably to cells conjugates exposed of U2OS track psoralen a Human along by 2007). localize to induced SLX4 human ICLs of ability ICL the of tested sites We to cells FA localize to in fails induction deletion UBZ containing SLX4 DISCUSSION AND RESULTS os Wlo ta. 03 eeldta noeosSX was SLX4 endogenous that revealed 2013) in- al., raised et panels). (Wilson antibodies lower 1B, house with (Fig. analysis TMP immunofluorescence to exposed Indirect been not had cells when B4tp oan aebe mlctdi recruiting in implicated been have domains UBZ4-type ti o la h amla L4hstoUZdomains UBZ two has SLX4 mammalian why clear not is It ˜oz 1 ai Wilson Jamie , 1 hmsJ MacArtney J. Thomas , c HA;n tiewsformed was stripe no -H2AX; 1 2811 ,

Journal of Cell Science HR REPORT SHORT 2812 data These S1A,B). SLX1 Fig. MUS81–EME1, material SLX4 (supplementary it XPF–ERCC1, C20ORF94 that of and and with 457/1–3, immunoblotting patients normally FA contrast, interacted from fibroblasts close-to- in By at levels expressed normal 1D). was SLX4 (Fig. that showed 457/ not immunoprecipitates and 457/2 did 457/1, DNA human patients PUVA-induced normal 3 from of fibroblasts in in tracks SLX4 SLX4 along damage, Whereas stripes ICLs. formed of fibroblasts sites to shown). recruitment of not stripe (data a stripe had SLX4 that (PUVA)-induced an cell displayed laser Every also psoralen–UVA 1C). (Fig. of damage sites DNA to recruited also et etse h oeo h L4UZdmisin domains UBZ SLX4 the of role the tested we Next, c -H2AX ugs hta es n ftetoSX B domains UBZ SLX4 two the leading sites, of damage recruitment. DNA SLX4 one at to protein least ubiquitylated a at recognizes that suggest rtisi hc osre ytiersde nete h is or first the either in first residues cysteine conserved Mutant (UBZ- we which beads. domains in amylose–agarose proteins UBZ and on the both immobilized containing were function, of 1+UBZ-2) SLX4 (MBP)- contribution of SLX4 protein fragments relative Maltose-binding fused to binding. the ubiquitin domains explore investigated UBZ to wished individual ubiquitin next to binds We domains UBZ SLX4 two polymers the of one Only ora fCl cec 21)17 8121 doi:10.1242/jcs.146167 2811–2817 127, (2014) Science Cell of Journal si n noeosSX oaiainwsaaye by 10 analyzed bars: was Scale localization immunofluorescence. SLX4 indirect endogenous using treated and were B fibroblasts in human patients normal as FA or from 457/3 SLX4 Cells and endogenous (D) 457/2 examined. of 457/1, was localization cells the U2OS except in B (endog.) for or As GFP (C) against H2AX. indirect antibodies to with subjected analysis and immunofluorescence UV-A fixed 355-nm were a Cells using laser. micro-irradiation subnuclear incubated 20 to were (TMP; subjected panels) trimethyl-psoralen (middle with only not) (upper GFP (or GFP–SLX4 or expressing panels) stably lower helix-turn- cells and HtH; U2OS motif: (B) PIAS motif. and helix Bric-a-brac Acinus domains Tramtrack, SAF-A/B, UBZ Broad-complex, SAP; BTB; SLX4 domain: first. tandem the black) the of in of part (outlined second and deletion the a of with all patients removing FA 457/2 are 457/1, 457/3 Siblings and nucleases. associated DNA and localized organization of sites PUVA. by to induced SLX4 damage of Recruitment 1. Fig. A iga hwn L4domain SLX4 showing Diagram (A) m ,6 i)and min) 60 M, m m. c -

Journal of Cell Science aawr bandwe h B a a elcdwith replaced was tag Similar MBP poly- effect. the no K63-linked when had to obtained UBZ-2 were binding mutating data – whereas abolished 2B). (Fig. alanine ubiquitin, polymers MBP-tagged UBZ-1 K63-linked K48-linked 2A). to to to (Fig. Mutating not binding but controls (UBZ-2*), robust mutated chains as poly-ubiquitin showed C336A+C339A used were (UBZ-1+UBZ-2) were SLX4 and domain – respectively (UBZ-1*) UBZ C296A+C299A second the REPORT SHORT er-bqii a uhwae.Teedt hwta SLX4 that or show tri- di-, data mono-, These Fig. to weaker. material ubiquitin Binding much (supplementary chains. six (UBZ-1+UBZ-2) was ubiquitin GST to to of tetra-ubiquitin SLX4 or bind 2C) to for minimum bind (Fig. S2C) MBP required A not to was S2A,B). fused did moieties Fig. binding, ubiquitin abolished alone material residues (supplementary GST–UBZ-2 cysteine conserved whereas and the poly-ubiquitin, K63-linked mutating to these bound with alone Consistent GST–UBZ-1 S2A,B). data, Fig. material (supplementary GST oanUZ1 u o B-,bnst oyuiutnchains K48-linkages. poly-ubiquitin over to K63- for binds preference UBZ-2, a not with but UBZ-1, domain ula oaiainsga n F a aal flclzn at localizing of capable a was both GFP to and fused in not alone signal cells data UBZ-1 localization the 3A; data, nuclear of (Fig. these any with micro-irradiated in Consistent been damage shown). had DNA that of mutant population sites SLX4-UBZ-1* the to the recruited but to not ICLs, recruited was PUVA-induced were (UBZ- of SLX4-UBZ-2* UBZ-2 sites and or the SLX4 (UBZ-1*) wild-type UBZ-1 Both or in 2*). GFP to mutations fused bearing SLX4 SLX4 wild-type containing viruses with infected os L4epesdi uieebyncfbolss(MEFs) fibroblasts of embryonic localization murine the from in on mutations expressed UBZ SLX4 of mouse effect the tested next RNF8, We FANCD2, domain, BRCA1 the UBZ-2 or requires the RAD18 damage not DNA but of domain, sites UBZ-1 to SLX4 of Recruitment Slx4 ora fCl cec 21)17 8121 doi:10.1242/jcs.146167 2811–2817 127, (2014) Science Cell of Journal 2 / 2 ie(atre l,2013). al., et (Castor mice 9 n y 9 nUZ1(rget2 rCs36and 336 Cys or 2) (fragment UBZ-1 Cys in at 299 Cys mutations and alanine 296 to cysteine assays. denote ubiquitin-binding Asterisks in used fragments domains SLX4 UBZ ubiquitin. SLX4 to the bind of ability to the Testing 2. Fig. niae eghwr sdwt rget 1–3. fragments with used mono- the were that of length except chains indicated B, poly-ubiquitin for K63-linked as or Same ubiquitin (C) performed blotting. membrane to the prior of staining panel Ponceau bottom a The shows antibodies. anti-ubiquitin immunoblotted with and (WB) SDS-PAGE to subjected Pulldowns were chains. 2–7) (pUb, or poly-ubiquitin on K48-linked K63-linked immobilized with were incubated alone and fragments MBP amylose–agarose The or (B) A 3). in (fragment shown UBZ-2 in 339 Cys A iga fteMBP-tagged the of Diagram (A) Slx4 2 / 2 Eswere MEFs 2813

Journal of Cell Science hc rmtshsoeuiutlto n erimn of recruitment RNF8, and 2010), FANCD2 al., ubiquitylation et promotes histone Song which 2010; promotes in al., implicated RAD18, which et been (Geng including have ligases ubiquitylation repair, E3 other ICL several ubiquitylated FANCI, be and not could 2001). al., that et FANCD2 (Garcia-Higuera 3B) K516R (Fig. mutant was a that FANCD2 manner wild-type or a expressing cells in PD20 vector, from empty indistinguishable with transfected cells human HR REPORT SHORT 2814 DNA of FANCD2 tracks in along treatment stripes PUVA normal by found form induced we light, could damage this ubiquitin SLX4 In for 2C). domain that with (Fig. UBZ-1 however, mono-ubiquitin SLX4 odds, over human at cells polymers the are DT-40 of data in preference These foci the 2011). subnuclear al., to et SLX4 SLX4 (Yamamoto chicken wild-type of containing recruitment cells in S3). than Fig. observed weaker material was stripes (supplementary stripes the of of intensity that the SLX4, average wild-type to on similar although damage, DNA PUVA-induced of sites eie h Acr ope htuiutltsFANCD2 ubiquitylates that complex core FA the Besides oouiutlto fFND a eotdt mediate to reported was FANCD2 of Mono-ubiquitylation 2 / 2 (PD20) osntrqieteE iae hthv led enimplicated been already have repair. that ICL ligases recruitment in E3 and ubiquitylated the cells of human require the sites in not FANCD2 but the does Taken not domain, to is 3C). question UBZ-1 recruited in (Fig. its is ligand intensity through SLX4 normal that damage show DNA of data stripe these SLX4 together, of an stripe a had with the cells, cell of also every UWB1.289 that cells; from 53BP1 HCT-116 (3) control indistinguishable parental was and relevant (2) BRCA1, prevented disrupted for null was S3), are that RAD18 which Fig. which shRNA in material cells RNF8 be (supplementary cells not an U2OS recruitment SLX4 (1) might expressing in 2012). protein damage Greenberg, PUVA-induced stably BRCA1, this and of and sites (Aressy of to 2012), repair recruitment activity al., DNA ligase et for required Yan E3 2012; the al., although et (Leung FAAP20 enx etdteefc fUZdmi uain nthe ICL on of mutations readout a domain as UBZ (MMC), C of mitomycin repair. to effect cells repair the of ICL for sensitivity tested required is next UBZ-2, not We but UBZ-1, domain SLX4 Slx4 ora fCl cec 21)17 8121 doi:10.1242/jcs.146167 2811–2817 127, (2014) Science Cell of Journal 2 N earadrcutett N aaesites. damage DNA to (A) to recruitment domains UBZ and SLX4 repair the DNA of contributions Relative 3. Fig. one,adrpeettv mgsaeson cl bars: Scale shown. are 10 images population, representative each and For counted, control. were a BRCA1 as expressing lacking used cells cells UWB1.289 UWB1.289 used. or were cells BRCA1 cells parental stably HCT-116 plus cells cells, RAD18 U2OS parental lacking that plus except shRNA B, RNF8 for expressing as Same (C) and analyzed. SLX4 laser Endogenous A. to in subjected as were micro-irradiation or panels), panels) (lower (middle K561R FANCD2 FANCD2 expressing stably cells PD20 or GFP c against indirect to antibodies subjected with and analysis fixed immunofluorescence a were using Cells laser. micro-irradiation UV-A subnuclear 355-nm to trimethyl-psoralen subjected with and incubated (TMP) were Cells or UBZ-2*. UBZ-1* SLX4 SLX4 (WT), wild-type (m)SLX4 mouse GFP-tagged HA.()P2 el akn AC2(pe aes,or panels), (upper FANCD2 lacking cells PD20 (B) -H2AX. / 2 m Slx4 m. el netdwt mt BB-uovector pBABE-puro empty with infected cells 2 / 2 Eswr netdwt ervrssexpressing retroviruses with infected were MEFs , 0 el were cells 300 c HA were -H2AX c -H2AX

Journal of Cell Science uatbhvdlk idtp L4(i.4) ossetwith Consistent 4A). (Fig. data, SLX4 these wild-type like behaved of sensitivity mutant capable MMC barely the was mutant rescuing UBZ-1* of SLX4 The SLX4. mouse type hs aaso htSX oanUZ1 u o B-,is UBZ-2, not but ICLs. UBZ-1, of SLX4. repair domain together, the SLX4 Taken for wild-type that vital 4B). show (Fig. radial data difference by whereas no these and rescue, made rescued prevented UBZ-2 completely breaks mutating UBZ-1 was domain as Mutating which (defined chromosomes), abnormalities chromosome netdwt h aevrs(i.4) utemr,teMMC the cells Furthermore, wild-type 4A). were (Fig. than virus of same hypersensitivity MMC the to with sensitive infected more much were REPORT SHORT xhne SE)idcdb M nMF Cso tal., et on mutations (Castor domain chromatid UBZ MEFs of sister effect in the the tested MMC of next we by third and 2013), one induced around (SCEs) for exchanges required of is resolution SLX4 the for required junctions are Holliday domains UBZ SLX4 Both Slx4 2 / 2 Slx4 Essoe nicesdicdneof incidence increased an showed MEFs 2 / 2 el a ece yutge wild- untagged by rescued was cells Slx4 2 / 2 el,btteUBZ-2 the but cells, eeeybutdin blunted severely ece hsdfc,btmttn ihrtefrto h second the data or These 4D). first (Fig. the rescue either prevented completely mutating domain but UBZ defect, this rescued rqec fSE nwl-yeMF yaon . fold. 2.5 around by MEFs the raises wild-type MEFs, MMC wild-type in 4C, with Fig. SCEs Compared in shown of As frequency SCEs. of occurrence the xrsigrtoiue eeue odpeeBMfrom BLM deplete (sh)RNA- the of to hairpin examined resolution used Short next for were context. We domains Slx4 this retroviruses 2013). UBZ in expressing al., SLX4 junctions et the (Castor Holliday Wyatt complex of BTR 2013; requirement the al., by dissolution et escape that junctions UBZ-1 was in mutations defect bearing SLX4 this 4C). of (Fig. but was version rescue the such MMC, with No SLX4-UBZ2*. observed with or SLX4 treatment wild-type by rescued after SCEs fewer i.S) elto fBMfo idtp Escue an caused MEFs wild-type from BLM of of increase Depletion S4). Fig. L4i eurdfrtenceltcrslto fHolliday of resolution nucleolytic the for required is SLX4 2 / 2 ora fCl cec 21)17 8121 doi:10.1242/jcs.146167 2811–2817 127, (2014) Science Cell of Journal Es(atre l,21)(upeetr material (supplementary 2013) al., et (Castor MEFs bomlte e eahs.W,wl-ye aaso the show Data of mean wild-type. number WT, the metaphase. were determine per spreads to abnormalities metaphase condition 50 each and for treated untreated, analyzed either left was or line MMC radial cell with of Each presence chromosomes. the ng/ml) for broken (20 analyzed and MMC and DAPI with with treated stained MEFs were of spreads Metaphase (B) i.4 eaiecnrbtoso h L4UZdmisto of domains junctions. analysis UBZ Holliday SLX4 of the resolution of the contributions Relative 4. Fig. eahs ped 10 nttl eeaaye.Data three analyzed. mean in were the chromosomes total) represent 400 in D, (1200 and spreads C the metaphase after In measured C, BLM. were for of MEFs as depletion in Same frequencies (D) SCE measured. that ng/ml) was expect (10 SCEs MMC of to frequency the exposed UBZ-1 and were domains (UBZ-2*) in UBZ-2 mutations or bearing (UBZ-1*) SLX4 or (WT) SLX4 otos aarpeettemean the represent Data controls. mSX,SX B-*o L4UZ2,epsdt the to exposed UBZ-2*, SLX4 or UBZ-1* SLX4 (m)SLX4, nrae el a eie s10.Wl-yeMF and MEFs Wild-type of 100%. viability as Slx4 the defined genotype, was each cells For untreated MMC. of doses indicated Slx4 , . odi C rqec u hsices was increase this but frequency SCE in fold 2.5 2 2 6 / / 2 2 ..()Wl-ye( Wild-type (C) s.d. Esifce ihepyvrs(vco)wr sdas used were (+vector) virus empty with infected MEFs Esifce ihrtoiue xrsigwild-type expressing retroviruses with infected MEFs Slx4 Slx4 2 2 / 2 / 2 6 Essal xrsigutge mouse untagged expressing stably MEFs s.e.m. Es(i.4) idtp SLX4 Wild-type 4D). (Fig. MEFs Slx4 Slx4 +/+ 2 MEFs, ) / 6 2 s.e.m., Esshowed MEFs A lngncsurvival Clonogenic (A) Slx4 n 5 2 3. / 2 Esand MEFs , 2815 30%

Journal of Cell Science CO bqii oyes eitsrcuteto L4t ie of domains sites to two to binds the SLX4 UBZ-2, of not analyzing recruitment but By mediates UBZ-1, recruited polymers, damage. that not ubiquitin is found DNA first, we the of of individually, part sites and domain all to UBZ lacks which second 457/1–3, the siblings German of of cells the for in dissolution. expressed required escape are that SLX4 junctions in Holliday domains of UBZ resolution the the of both that suggest REPORT SHORT rait rc ln elnce.Tepwro h ae i em of terms (in laser to the used of 2816 was power (Zeiss) The nuclei. microscope cell PALM with along a track incubated to a attached were irradiate laser dishes UV-A glass-bottomed nm (25 35-mm [TMP in trimethyl-psoralen seeded microscopy Cells confocal and irradiation Laser DMEM and in pyruvate sodium grown 37 with (w/v)], at supplemented [1% acids were (FBS) amino streptomycin non-essential serum (w/v)], bovine (MEFs) [1% fetal penicillin 10% fibroblasts with supplemented embryonic Mouse lines Cell METHODS AND investigation. MATERIALS SLX4 of the area of interesting both an for to be ligands SLX4 will the recruits domains Finding that UBZ damage. protein the DNA linkages in of K33 domain sites or ubiquitin-like K27 a K11, to as or such chains poly-ubiquitin atypical chains poly-ubiquitin K63-linked bind vitro or not in K48- does have to it to binding, or ubiquitin mono-ubiquitin appears for to UBZ-2 necessary though repair, residues the Even ICL of idea. for all not this but with Holliday BTR, consistent of by is resolution dissolution the escape for that required present junctions is the UBZ-2 in SLX4 observation that The study 2013). al., repair et DNA in and branched (Castor complex alternative intermediates cleavage of SLX4 junction cleavage the Holliday the of reflects of Recent probably role independent the complex. is that Holliday ICLs BTR suggests repairing of the lab our resolution by from dissolution the data escape for that UBZ-1, junctions with together required, involved, previously ligases ligase E3 repair. E3 the ICL of relevant in any from the implicated distinct to as be interesting to well be appears will as which It protein ICLs. poly-ubiquitylated of this a sites identify to poly- exists SLX4 there recruits not that that ligand mono-ubiquitylated, predict is ubiquitin We six ubiquitylated. FANCD2 least that at observation whereas of our chains Song moieties, with poly-ubiquitin 2010; to consistent binds is are al., only which data SLX4 et These RAD18, 2010). (Geng lacking al., ubiquitylation cells et FANCD2 in in to normally and for SLX4 occurs FANCD2 required of manner lack recruitment this that in the induced cells that damage found 2011). DNA we al., of sites et study, (Yamamoto present in MMC foci the of to In formation exposed the cells for chicken required of DT-40 be form to reported mono-ubiquitylated was The FANCD2 UBZ-1? through SLX4 recruits 457/1-3. SLX4 patients target in correctly essential to phenotype failure be FA that the to likely is explains appears it and UBZ-1, repair, by ICL proper for mediated Therefore, SLX4, efficient MEFs. in of both ICLs by targeting for induced essential SCEs and is repair ICL and damage DNA PUVA-induced eimsplmne ih1%FS %pncli-tetmcnand penicillin-streptomycin 1% DMEM glutamine. in FBS, mM cultured 10% 2 and with SV-40 with supplemented immortalized medium were 457/3 and 457/2 lhuhSX B- sdsesbefrILrpi,i is it repair, ICL for dispensable is UBZ-2 SLX4 Although that damage DNA of sites at ligand ubiquitylated the is What SLX4 of form mutated the that showed we study, this In 2 omlhmnfbolsso irbat rmF ains457/1, patients FA from fibroblasts or fibroblasts human Normal . Fg ) ti osbe oee,ta hsdmi id to binds domain this that however, possible, is It 2). (Fig. m ) im-lrc]fr6 i.A355- A min. 60 for Sigma-Aldrich] M); ˚ nahmdfe topeeudr5% under atmosphere humidified a in C Mun odvn .L n ’nra .D. A. D’Andrea, and L. G. Moldovan, K. Hofmann, Geng,L.,Huntoon,C.J.andKarnitz,L.M. ecn nest)wsstt 0 ognrt Cs n h ra were 40 areas Fluor the Plan a and using ICLs, by targeted generate 3 was to A speed. 20% low to at struck set was intensity) percent uhrcontributions Author interests. competing no declare authors The interests Competing RAD18. lacking cells HCT-116 for for Japan) Center Chiba, (Research Therapy, Shiomi Particle Tadahiro Charged and antibodies, U2OS RNF8 providing and for cells Denmark) shRNF8 Copenhagen, Research, Protein for of (both Center Mailand Niels the and Bekker-Jensen Simon thank We microscopy. to with grateful help for are We Na Inke cells. and PD20 Appleton for Paul MA) Cancer Boston, (Dana-Farber D’Andrea School, Alan Medical thank Harvard Institute, We study. fibroblasts, this donating for for important 457/3 were and which 457/2 457/1, patients to grateful sincerely are We Acknowledgements previously described 2013). were al., et abnormalities (Castor previously SCE chromosome of described analysis and and SLX4 as mouse frequencies against Antibodies out 2013). al., carried et (Wilson was immunofluorescence Indirect Miscellaneous en,J . ag . og .W,He,M . i .adCe,J. Chen, and L. and Li, D. S., M. A. Huen, W., Auerbach, K. Fong, H., Y., Wang, Hanenberg, W., J. R., Leung, Desetty, P., F. Lach, Y., Kim, C., Timmers, S., M. Meyn, S., Ganesan, T., Taniguchi, I., Garcia-Higuera, S., Coulon, A., Tissier, R., E. Taylor, C., Chahwan, S., J., Scaglione, T. S., Macartney, Fekairi, R., Toth, C., Lachaud, C., A. Declais, N., Nair, D., Castor, A. R. Greenberg, and B. Aressy, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.146167/-/DC1 online available material Supplementary material Supplementary release. Unit. immediate Ubiquitylation for and PMC Phosphorylation in Protein Deposited Pfizer) MRC and the Pharmaceutica with Boehringer- Janssen associated (AstraZeneca, KgaA, Unit Merck Therapy the GlaxoSmithKline, Transduction by Ingelheim, Signal supported supporting companies of also pharmaceutical Division was the the study and This (MRC); Fellow. Council Clinical Research and Trust Medical (FanDamAge); Wellcome fellowship a Intra-European Curie is Marie J.S.J. a of recipient the is C.L. Funding provided D.S. 457/1–3. constructs; patients FA cDNA from made T.J.M. fibroblasts MEFs mutants; Slx4-null UBZ provided with J.W. complemented immunoprecipitations; I.M. experiments; SLX4 ubiquitin-binding D.C. with the experiments; helped performed the K.H. of analyses; majority SCE the with performed helped C.L. and study the conceived J.R. efre iiu ftretms n iiu f10clswere cells 100 of minimum was a replicate. and per experiment times, treated Each three PUVA of immunofluorescence. minimum a indirect performed to subjected then uio . ecmn,J . oh . aate,T,Epn,B tal. et B. Eppink, T., Macartney, R., Toth, M., J. Heuckmann, L., Pulido, genome. the guards network. repair DNA anemia Fanconi the Biol. activates Cell J. PCNA of ubiquitination ru FNA n atcptsi nesrn rs-ikrepair. cross-link interstrand complementation in FA USA participates Sci. stabilizes and Acad. FAAP20 (FANCA) protein A binding group (FA) anemia Fanconi Genet. Nat. A. Smogorzewska, response. pathway. repair/ common a DNA in D. BRCA1 multiple A. and D’Andrea, proteins binds and anemia M. that Grompe, J., subunit Hejna, resolvase al. junction et P. endonucleases. Russell, Holliday recombination III, R., a J. Yates, C., is Ruse, Q., M. Dong, Nucleases. MUS81-EME1 and SLX1 the by Cell repair Mol. DNA J. and Rouse, resolution and junction S. J. Arthur, M., D. Lilley, context. proper z .M,Hi,K,De K., Hain, M., I. oz, ˜ ora fCl cec 21)17 8121 doi:10.1242/jcs.146167 2811–2817 127, (2014) Science Cell of Journal 52 N ear(Amst.) Repair DNA 20) bqii-idn oan n hi oei h N damage DNA the in role their and domains Ubiquitin-binding (2009). 221-233. , 191 43 142-146. , ur Biol. Curr. 249-257. , 109 4491-4496. , nu e.Genet. Rev. 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Journal of Cell Science aiuh,T,Gri-iur,I,Adese,P . rgr,R . Grompe, C., R. Gregory, R., P. Andreassen, I., Garcia-Higuera, T., Taniguchi, P., S. Gygi, C., B. O’Connell, E., M. Sowa, A., Smogorzewska, M., J. Svendsen, tekr . an . cutr . ihrtHfte . oias .A., M. Rooimans, Y., Hilhorst-Hofstee, B., C. Schuster, Vaziri, K., and Hain, C., M. Stoepker, G. Kupfer, S., Tateishi, A., Gurkar, K., Palle, Y., I. Song, HR REPORT SHORT leg,S .adHre,J W. J. Harper, and J. S. Elledge, nmapoen AC2 ihBC1adRAD51. and BRCA1 with D. FANCD2, protein, A. anemia repair. D’Andrea, DNA and for M. required is and resolvase 77. junction Holliday a 21) L4 oriao fsrcueseii noulae,i uae na al. in et subtype. mutated W. is anemia A. endonucleases, Nieuwint, Fanconi T., structure-specific new E. of Korthof, coordinator K., a Eirich, SLX4, (2011). B., A. Oostra, J., Steltenpool, pathway. repair DNA anemia Fanconi coupled the is 31525-31536. adducts of DNA activation bulky of to synthesis translesion Rad18-mediated (2010). eurdfrDArepair. is SLX4/BTBD12 DNA human for by required nucleases structure-specific of Coordination (2009). o.Cell Mol. 20) -hs-pcfcitrcino h Fanconi the of interaction S-phase-specific (2002). a.Genet. Nat. 20) amla TD2SX assembles BTBD12/SLX4 Mammalian (2009). 35 116-127. , 43 138-141. , Blood .Bo.Chem. Biol. J. 100 2414-2420. , Cell 138 63- , 285 , a,Z,Go . aaaia,M,Se,W,Ln,C,Fx . I,Wn,Y., Wang, III, D., Fox, C., Ling, W., Shen, M., Paramasivam, R., Guo, Z., Yan, Kono, M., Takata, H., C. Kurumizaka, M., S. Tsuda, S., West, Kobayashi, N., K. and Yamamoto, J. Matos, S., Sarbajna, D., H. Wyatt, isn .S,Tjr,A . atr . oh . lso .A n os,J. Rouse, and A. M. Blasco, R., Toth, D., Castor, M., A. Tejera, S., J. Wilson, hzahetl .K,Lu .T,Idg .E n eda,M M. M. Seidman, and E. F. Indig, T., S. Liu, K., A. Thazhathveetil, AP0 ik N8mdae bqiiaint h acn nmaDArepair al. DNA anemia et Fanconi Y. the D. network. to ubiquitination Lee, RNF8-mediated J., links Kuehl, FAAP20, B., A. Oostra, pathway. anemia K. Fanconi USA the Hirota, Sci. by Acad. and regulated Natl. is S. repair Takeda, cross-link interstrand in J., resolution Jiricny, junction K., Holliday for MUS81-EME1 and cells. human SLX1-SLX4 of actions 21) oaiaindpnetad-needn oe fSX nregulating in SLX4 of roles -independent telomeres. and Localization-dependent (2013). srlncnuae o iulzto fgnmcitrtadcross-links interstrand genomic of photoactivation. visualization laser by for localized conjugates Psoralen ora fCl cec 21)17 8121 doi:10.1242/jcs.146167 2811–2817 127, (2014) Science Cell of Journal o.Cell Mol. elRep. Cell o.Cell Mol. 47 4 61-75. , 108 853-860. , 52 6492-6496. , 234-247. , icnu.Chem. Bioconjug. 21) bqii-idn protein, ubiquitin-binding A (2012). 21) novmn fSX in SLX4 of Involvement (2011). 18 431-437. , 21) Coordinated (2013). (2007). 2817 Proc.

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