Hsapiens H. 47 ALFVQFNDQFFWGQLEAVEVKWSVRMTLCAGICSYE---GKGGMCSIRLSEPLLKLRPRK

Transcription

1 Supplementary Figures and Figure Legends Supplementary Figure 1 * * FAN1 KLACPVCSKMVPRYD...LNRHLDEMCANNDF POLK ILTCPVCFRAQGCISLEALNKHVD.ECLDGPS ALVCPVCNVEQKTSDLTLFNVHVD.VCLNKSF RAD18 KVDCPVCGVNIPESH...INKHLD.SCLSREE RNF220 GQVCPLCNRPLAGSE.QEMSRHVE.HCLSKRE UIMC1 QVSCPLCDQCFPPTK...IERHAM.YCNGLME NEKCYLCKSLVPFRE...YQCHVD.SCLQLAK WRNIP QVQCPVCQQMMPAAH...INSHLD.RCLLLHP ZBTB1 PFRCPNCGQRFETENL..VVEHMS.SCLD... ZNRF1 GFKCPICSKSVASDE...MEMHFI.MCLSKPR ZNRF2 GFKCPVCSKFVSSDE...MDLHLV.MCLTKPR SLX4 LFFCQICQKNLSAMNVTRREQHVN.RCLDEAE IPECPICGKPFLTLKS..RTSHLK.QCAVKME C1ORF124 MVNCPVCQNEVLESQ...INEHLD.WCLEGDS DCLRE1A DGYCPNCQMPFSSLIGQTPRWHVF.ECLDSPP FAM164A LLPCKICGRTFFPV..LKKHGP.ICQKTAT YIQCPYCQRRFNEN..ADRHIN.FCKEQAA FAM164B LFPCEVCGRRFAADV...LERHGP.ICKKLFN FAM164C LGECSHCGRKFLSFR...LERHSN.ICSRMRG RNF220 ZBTB1 ZNRF1 ZNRF2 FAM164A FAM164C GFP GFP + DAPI Hsapiens Btaurus Xlaevis Ggallus Celegans Hsapiens Btaurus Xlaevis Ggallus Celegans MDDDLMLALRLQEEWNLQEAER-----D-HAQESLSLVDASWELVDPTPDLQ MDEDLVLALQLQEEWNLQVSER-----E-PAQEPLSLVDASWELVDPTPDLQ MDEDLVVALRLQEEWDVQMARR-----AAAAREPVSLVDASWELVDPTPDLQ MQLSVVDPTWELLDPNPDIR 1 MEQEMQDEEDDDFALALQLQELWEAEAAAGVEAPPDPASLRPLSVVDEAWELLDPSPDVH MSRSSLVDPSFELSDPCPDIH 47 ALFVQFNDQFFWGQLEAVEVKWSVRMTLCAGICSYE---GKGGMCSIRLSEPLLKLRPRK 47 GLFVLFNDRFFWGQLEAVEVKWSVRMTLCAGICSYE---GRGGMCSIRLSEPLLKLRPRK 48 ALFLQFNDRFFWGQLEAVEVKWSVRMTLCAGICTYE---GRGGMCSIRLSEPLLKLRPRK 21 ALFLEFNDTFFWGQLSGIEVKWSARMTLCAGVCSYE---GRGGLCSIRLSEPLLKLRPRK 61 GLFVQFNEALFWGRLAAVEVSWSPRMTLCAGVCKYK---GNGGMCSIRLSEPLLKLRPRK 22 ALFIQFDARFFGGSLACCEVKWSPRMYACAGICSYEIRGGRGGLCSIRLSKPLLTLRPRS E. C. H. sapiens taurus D. M. musculus X. laevis G. gallus C1ORF124 () Hsapiens Btaurus Xlaevis Ggallus Celegans 104 DLVETLLHEMIHAYLFVTNNDKDREGHGPEFCKHMHRINSLTGANITVYHTFHDEVDEYR 104 DLVETLLHEMIHAYLFVTNNDKDREGHGPEFCKHMHRINRLTGANITVYHTFHDEVDEYR 105 DLVETLLHEMIHAYLFVTNNDKDREGHGPEFCKHMHRINQLTGANITVYHTFHDEVDEYR 78 DLVQTLLHEMIHALLFVTHNNKDHDSHGPEFCKHMERINKRTGANISVYHNFHDEVDEYR 118 DLVETLLHEMIHALLFVTNNDRDRDSHGPEFCKHMRRINRLTGANVTIYHNFHDEVDSYR 82 DLVETLLHEMIHAYLFVKERNRDRDGHGPQFQAHMHRINQAGGTNITIYHSFHDEVRLYK SprT-like domain PIP box Hsapiens 164 RHWWRCNGPCQHRPPYYGYVKRATNREPSAHDYWWAEHQKTCGGTYIKIKEPENYSKKGK Btaurus 164 RHWWRCNGPCQNSKPYYGYVKRATNRAPSAHDYWWAEHQKTCGGTYIKIKEPENYSKKGK 165 RHWWRCNGPCQHRQPYYGYVKRATNRAPSVHDYWWADHQKTCGGTYIKIKEPENYSKKGR Xlaevis 138 KHWWLCNGPCQKRKPYFGYVKRAMNRAPSSLDPWWADHQRTCGGEFVKIKEPENYSQKRK Ggallus 178 QHWWRCNGPCQYRRPYFGYVKRSMNRAPSARDFWWAEHQENCGGTFTKVKEPENFSKKPK H. sapiens taurus M. musculus X. laevis G. gallus C. elegans MDDDLMLALRLQEEWNLQEAER-----D-HAQESLSLVDASWELVDPTPDLQ Celegans 142 QHWWRCSGPCRDRRPFFGYVKRSCNRAPGPNDRWWSQHQQSCGGNFLKVKEPEGYGQGKG MDEDLVLALQLQEEWNLQVSER-----E-PAQEPLSLVDASWELVDPTPDLQ MDEDLVVALRLQEEWDVQMARR-----AAAAREPVSLVDASWELVDPTPDLQ MQLSVVDPTWELLDPNPDIR Hsapiens 224 GKAKLGKEPV----LAAENKDKPNRGEAQLVIPFSGKGYVLGETSNLPSPGKLITSHAIN 1 MEQEMQDEEDDDFALALQLQELWEAEAAAGVEAPPDPASLRPLSVVDEAWELLDPSPDVH Btaurus 224 GKTKLRKQPV----SEAENKDKPNRGEKQLLIPFTGKGYVLGETSNFS-SGKCITSHAIN MSRSSLVDPSFELSDPCPDIH 225 GKTKADKQPA----SAVENKDKLCRGEAQLLIPFSGKGYVLGDASTCPSAGKLNTSYMVN Xlaevis 198 RNNDPTKSELGNSSHVKINKGKSNGVDIRTVIPFSGTGYKLFEPSKSDAQLKIQNDN--- Ggallus 238 EKTETEKLQN----SKSTNKGQVHLGDKKDPKPFSGNGYRLGGAGSGLSEKSVTFSSNVM Hsapiens H. 47 ALFVQFNDQFFWGQLEAVEVKWSVRMTLCAGICSYE---GKGGMCSIRLSEPLLKLRPRK Celegans 202 SKRTNDKNKSGGPALKKTI--TPPRVTLDD--FFKKDGKNSSDNSTSKSPTKP Btaurus taurus 47 GLFVLFNDRFFWGQLEAVEVKWSVRMTLCAGICSYE---GRGGMCSIRLSEPLLKLRPRK M. 48 ALFLQFNDRFFWGQLEAVEVKWSVRMTLCAGICTYE---GRGGMCSIRLSEPLLKLRPRK Xlaevis X. laevis 21 ALFLEFNDTFFWGQLSGIEVKWSARMTLCAGVCSYE---GRGGLCSIRLSEPLLKLRPRK Hsapiens 280 KTQDLLNQNHSANAVRPNSKIKVKFEQNGSSKNSHLVSPAVS NSHQNVLSNY Ggallus G. gallus 61 GLFVQFNEALFWGRLAAVEVSWSPRMTLCAGVCKYK---GNGGMCSIRLSEPLLKLRPRK Btaurus 279 ESQEPLSQDHSANALRPHSKTEVKFEQNGPSKKTSVASPVLS TSHQNVLSNY Celegans C. 22 ALFIQFDARFFGGSLACCEVKWSPRMYACAGICSYEIRGGRGGLCSIRLSKPLLTLRPRS 281 EAKGLSSQDHSVSGLRLNSNAEVKCEQNCLPKKPHLVSPLPT ASHQSVLSSY Xlaevis 255 PTKDKAVMHH T PPSTNQT----DSTFLSRKIVS Ggallus ** * 294 NTGRSDSQHRSAGSTIPIPKNEIKFEKSPRHDT---FFPLFTGDA----SEKMNLASKRE Hsapiens H. 104 DLVETLLHEMIHAYLFVTNNDKDREGHGPEFCKHMHRINSLTGANITVYHTFHDEVDEYR Celegans ST----SLFTGSGQKLGGSSSTSSLLNS Btaurus taurus 104 DLVETLLHEMIHAYLFVTNNDKDREGHGPEFCKHMHRINRLTGANITVYHTFHDEVDEYR M. 105 DLVETLLHEMIHAYLFVTNNDKDREGHGPEFCKHMHRINQLTGANITVYHTFHDEVDEYR Xlaevis X. laevis 78 DLVQTLLHEMIHALLFVTHNNKDHDSHGPEFCKHMERINKRTGANISVYHNFHDEVDEYR Hsapiens 332 FPRVSFANQKAFRGVNGSPRISVTVGNIPKNSVSSSSQRRVSSSKISLRNSSKVTESASV Ggallus G. gallus 118 DLVETLLHEMIHALLFVTNNDRDRDSHGPEFCKHMRRINRLTGANVTIYHNFHDEVDSYR Btaurus 331 FSKVSVASSKAFRSVSGSPVKSLTVGDSTTKSVSAGSQRRVTSSRTSLRNSLKAMESTYV Celegans C. 82 DLVETLLHEMIHAYLFVKERNRDRDGHGPQFQAHMHRINQAGGTNITIYHSFHDEVRLYK 333 FPRVSVANQKAFRNVNGSPVKNGTTGDGTKRPASGGSQRKVPPSRASLRNTSKVTAPASA Xlaevis 284 AKKISVANTKVFINLNGSPIKLPSSSNNKSHQDSSKQKSVLHFFKTQKDNSIDLTSSSQS Ggallus 347 FPKLSVANTKAYKNVNGSPVKVARVVEEDSNQGSAKGKRVLPPCYNRSPKQVRFQQTAAA Hsapiens H. 164 RHWWRCNGPCQHRPPYYGYVKRATNREPSAHDYWWAEHQKTCGGTYIKIKEPENYSKKGK Celegans 2 YPK ATQNSGGNRLGGTSGGVSRLLPPVNFTSPSSAPVAEQVI- Btaurus taurus 164 RHWWRCNGPCQNSKPYYGYVKRATNRAPSAHDYWWAEHQKTCGGTYIKIKEPENYSKKGK M. 165 RHWWRCNGPCQHRQPYYGYVKRATNRAPSVHDYWWADHQKTCGGTYIKIKEPENYSKKGR Xlaevis X. laevis 138 KHWWLCNGPCQKRKPYFGYVKRAMNRAPSSLDPWWADHQRTCGGEFVKIKEPENYSQKRK Hsapiens MPSQDVSGSEDTFPNKRPRLEDKTVFDNFFIKKEQIKSSGNDPKYST--- Ggallus G. gallus 178 QHWWRCNGPCQYRRPYFGYVKRSMNRAPSARDFWWAEHQENCGGTFTKVKEPENFSKKPK Btaurus TVPQDAGGPEGKLPSKRPRIEDKTFFDLFFIKKEQAQSGGGDVTSSSHP- Celegans C. 142 QHWWRCSGPCRDRRPFFGYVKRSCNRAPGPNDRWWSQHQQSCGGNFLKVKEPEGYGQGKG 393 TVTSAAGTSATISREESGSENQFLNKRPRLEDRTALDTI---KEQTQS-GGDLQSSS-Q- Xlaevis FPSTSQGPNREETEHFYKKLQMDDKESKDTFIIHSLNKTNVSDSLNNKS--- Ggallus QTP-SEKKSLENTVQQWPRKEDQTTFENYFIKKHPDVTSSINTPLKTKAE Hsapiens 224 GKAKLGKEPV----LAAENKDKPNRGEAQLVIPFSGKGYVLGETSNLPSPGKLITSHAIN Celegans DLGDSDDDDF---QDMD Btaurus 224 GKTKLRKQPV----SEAENKDKPNRGEKQLLIPFTGKGYVLGETSNFS-SGKCITSHAIN * * ** 225 GKTKADKQPA----SAVENKDKLCRGEAQLLIPFSGKGYVLGDASTCPSAGKLNTSYMVN * * 324 Xlaevis QNVLSNYF 198 RNNDPTKSELGNSSHVKINKGKSNGVDIRTVIPFSGTGYKLFEPSKSDAQLKIQNDN--- Hsapiens H TTAQNSSSSSSQSKMVNCPVCQNEVLESQINEHLDWCLEGDSIKVKSEESL 323 Ggallus QNVLSNYF 238 EKTETEKLQN----SKSTNKGQVHLGDKKDPKPFSGNGYRLGGAGSGLSEKSVTFSSNVM Btaurus taurus F PAAAQSPSGASGQSRVVHCPVCQDEVSETQINEHLDWCLERDSTQVKS---- Celegans 202 SKRTNDKNKSGGPALKKTI--TPPRVTLDD--FFKKDGKNSSDNSTSKSPTKP M. 325 QSVLSSYF 447 -PTAASAPQSLSSQRRLVNCPVCQGVVVESQINEHLDRCLEGNKTNLRPRRV- Xlaevis X. laevis 393 -CAGPAATINSGLNHTKVCCPVCGTEIFESKINDHLDTCLQNYNT QKSVLPFF Ggallus G. gallus 456 SAVCSPRSSTAERQEKTVSCPVCQTEVLESKINEHLDSCLV Hsapiens QTTFENYF 280 KTQDLLNQNHSANAVRPNSKIKVKFEQNGSSKNSHLVSPAVS NSHQNVLSNY Celegans C DDALEISFVASDNSVICPSCNTEVMENLIHGHLDYCLG Btaurus 279 ESQEPLSQDHSANALRPHSKTEVKFEQNGPSKKTSVASPVLS TSHQNVLSNY 281 EAKGLSSQDHSVSGLRLNSNAEVKCEQNCLPKKPHLVSPLPT ASHQSVLSSY UBZ

2 Supplementary Figure 1. Alignment of C1ORF124 domains (a) Alignment UBZ4 domains from human proteins. Identical residues are shaded in black, similar residues are shaded in grey. The conserved Cys and His residues that define the two dyads of the UBZ domain are shaded in red. The asterisk denotes the conserved Cys residues in the first dyad; these correspond to Cys456 and Cys459 in C1ORF124 (). (b) Fluorescence analysis of U2OS cells, transiently expressing uncharacterized UBZ4 domain-containing proteins (C1ORF124, RNF220, ZBTB1, ZNRF1, ZNRF2, FAM164A, FAM164C) fused to GFP. (c) Schematic diagram of the domain organization of C1ORF124 (). (d-f) Multiple sequence alignments of the SprT-like domain (d), PCNAinteracting protein (PIP) box motif (e) and UBZ domain (f) of. Asterisks in (d) denote conserved metallopeptidase motif in the SprT-like domain. Asterisks in (e) denote residues Gln325, Leu328, Tyr331 and Phe332 in the PIP box. Asterisks in (f) denote conserved residues Cys456 and Cys459 in the UBZ domain.

3 Supplementary Figure 2 Time (h): Tetracycline (µg/ml) GFP- RNF220 ZNRF1 ZNRF2 FAM164A FAM164C GFP + MMC GFP + DAPI C. Time (min): GFP- ZBTB D. 100 GFP- Wild type CCAA GFP- GFP- FAN1 Supplementary Figure 2. Generation of stable cell lines (a) Western blot analysis of U2OS Flp In cells stably expressing GFP- after incubation with tetracycline at the indicated concentration for 3 and 12h. (b) Fluorescence analysis of U2OS cells transiently expressing UBZ4 domaincontaining proteins fused to GFP, treated with mitomycin-c (MMC, 300 ng/ml) for 18 h, followed by fixation. (c) Live cell fluorescence imaging of U2OS cells, transiently expressing GFP-ZBTB1 or GFP-FAN1, at the times indicated after laser micro-irradiation. (d) Western blot analysis of U2OS Flp In cells stably expressing GFP--WT or GFP--CCAA (C456A, C459A).

4 Supplementary Figure 3 GST pulldown GST GST- GST-CCAA GST-PIP Blot: GST Blot: PCNA parental HCT-116 Rad18 -/- sirna: Time after UV (h): control Long exposure Short exposure Mono-Ub- PCNA PCNA PCNA Supplementary Figure 3. is not required for PCNA ubiquitylation (a) GST pulldown analysis of recombinant GST- fusion proteins ( wild type, UBZ mutant (CCAA; Cys456, Cys459) or PIP box mutant (PIP*; Gln325, Leu328, Tyr331, Phe332) expressed in bacteria, immobilized on glutathionesepharose and incubated with extracts of U2OS cells that were irradiated with UV-C (254 nm; 2 Jm -2 ) and allowed to recover for 6 h. (b) Western blot analysis of PCNA ubiquitylation in parental HCT-116 cells treated with control sirna or -specific sirna (#17), and HCT-116 Rad18 -/- cells. Cells were exposed to UV-C irradiation (254 nm; 2 Jm -2 ) and allowed to recover for the times indicated. Cells were lysed in RIPA buffer and after centrifugation at 13,500 rpm; the supernatant (S1) was placed on ice and the chromatin pellets were resuspended in RIPA buffer supplemented with magnesium chloride (5 mm) and benzonase (50 units). The re-suspended pellets were centrifuged at 13,500 rpm and the resulting supernatant (S2) was combined with supernatant S1. The combined supernatants were subjected to western with antibodies against PCN The migration positions of PCNA and mono-ubiquitylated PCNA are indicated.

5 Supplementary Figure 4 sirna Control #17 #19 50 FLAG-p97 + GFP--wild type FLAG-p97 + GFP--CCAA FLAG-p97 + GFP FLAG + GFP--wild type FLAG + GFP--CCAA ip: anti-:flag ip: anti-:gfp Blot: FLAG Blot: GFP Blot: FLAG Blot: GFP Supplementary Figure 4. The UBZ and PIP motifs are not required for p97 interaction (a) Western blot analysis of U2OS cells transfected with the indicated - specific sirnas for 48 h. (b) Western blot analysis of anti-flag and anti-gfp immunoprecipitates of cells transiently expressing FLAG-p97 and GFP-, or the relevant empty vectors.

6 Supplementary Figure 5 Time (min): sirna + mcherry--ccaa mcherry- -WT Merge - CCAA GFP-p97 Merge - Wild type GFP-p97 Supplementary Figure 5. Live imaging and p97 at sites of DNA damage Live cell fluorescence imaging of U2OS cells, treated with -specific sirna (#17 and #19), transiently expressing GFP-p97, and mcherry- WT or CCAA plasmids that were resistant to the sirnas, at the times indicated after laser-micro-irradiation.

7 Supplementary Figure 6 Control sirna #17 #19 50 GFP-Pol GFP-Pol +DAPI UV sirna C. Control NPL4 + NPL4 NPL4 37 Supplementary Figure 6. Knockdown of and analysis of Pol η foci (a) Western blot analysis of U2OS cells transfected with the indicated - specific sirnas for 48 h. (b) Immunofluorescence analysis of U2OS cells transiently expressing GFP-Pol η after UV-C irradiation (254 nm; 2 Jm -2 ) and allowed to recover for 6 h. (c) Western blot analysis of U2OS cells transfected with the indicated a -specific sirna (#17), an NPL4-specifc sirna, or both sirnas for 48 h.

8 Supplementary Figure 7 SprT-like domain SHP PIP box UBZ SHP box FXGXGQRU -H.sapiens 252 FSGKGYVL -taurus 252 FTGKGYVL -M.musculus 253 FSGKGYVL -X.laevis 230 FSGTGYKL -G.gallus 266 FSGNGYRL Supplementary Figure 7. has an SHP box (a) Schematic diagram of the domain organization of including the SHP box. (b) Multiple sequence alignment of the SHP box.

9 Supplementary Table 1 Protein Name Access Expected Mass (Da) Mascot Score No. of peptides HUWE1 Q7Z6Z MKI67 P PCM1 Q NISCH Q9Y2I MYBBP1A Q9BQG SMC3 Q9UQE IRS4 O VCP P SFPQ P HSPA9 P WRNIP1 Q96S HSPA5 P NPLOC4 Q8TAT BAG3 O TCP1 P CCT5 P PKM2 P CCT4 P CCT2 P C1orf124 Q9H EEF1A1P5 Q5VTE EEF1A1 P PSMD11 O ENO1 P FAM1B Q PSMD6 Q PSMC5 P DNAJA1 P NSFL1C Q9UNZ HSPA7 P CSDA P HSP90AA5P Q58FG YBX1 P STUB1 Q9UNE UFD1L Q SLC25A1 P HAGH Q FBL P SLC25A4 P SLC25A6 P SLC25A6 P SLC25A5 P ATP5C1 P TPM3 P PGAM5 Q96HS C1QBP Q PRDX4 Q NDUFS3 O PHB P PSMA1 P PSMA4 P YWHAE P PCNA P EMD P PGAM1 P PSMB5 P SNRPA1 P Supplementary Table 1. Identification of -interacting proteins Extracts of HEK293 cells stably transfected with GFP-, or GFP only, were subjected to immunoprecipitation with GFP-Trap beads. After SDS PAGE (Fig. 5A), the relevant gel lanes were cut into slices and proteins identified by mass fingerprinting. Proteins identified in GFP- precipitates that were not in GFP only precipitates are shown.