Mutations of the SLX4 Gene in Fanconi Anemia

© 2011 Nature America, Inc. All rights reserved. bone genome Fanconi Agata Smogorzewska Yonghwan Kim Mutations of the s r e t t e l 142 Received 31 August 2010; accepted 15 December 2010; published online 16 January 2011; to this work. should Correspondence be addressed to A.S. ([email protected]). Heine University, Duesseldorf, Germany. Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indiana, Indianapolis, USA. 1 other The FANCD2. of monoubiquitination lack FANCM) (except for complex core anemia Fanconi the in mutations with Cells inte 9,10). repair direct and FANCI- (refs. chromatin an to as complex FANCD2 FANCD2 localize and subsequently proteins FANCI activated two These ubiquitinates which ligase ubiquitin E3 nuclear a FANCG, complex, FANCF, core a form FANCM) and FANCL FANCE, FANCC, FANCB, (FANCA, proteins anemia Fanconi for responsible anemia Fanconi thecin campto to and agents crosslinking to sensitivity increased to leads (ICLs) crosslinks interstrand of processing the in implicated nucleases, previously MUS81 been have which and of both ERCC4/XPF of activity the stimulate also can SLX4 complex, the in seen activity resolvase junction the Holliday for responsible largely is interaction SLX4-SLX1 the Although MUS81-EME1 and ERCC4/XPF-ERCC1 SLX1, nucleases: tinct dis three with interacts that protein scaffold is a multidomain SLX4 subtype mutations complemented and two Here in recently SLX4, crosslinked the Fanconi feature hypersensitivity malignancies Laboratory Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, USA.

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dinucleotide GT in intron 5 (c.1163+2T>A) in the genomic DNA genomic ( the in (c.1163+2T>A) 5 intron in GT dinucleotide donor splice canonical the in mutation point homozygous a to due individual’s cells revealed skipping of exon 5 ( ( inhibitor I topoisomerase a ingly, not individual 1084/1 (RA3083) were sensitive to camptothecin, Fig. in sensitivity fibr ultraviolet no observed We MMC. to sensitivity and FANCD2 mon FANCD2 FANCN indiv disorder three anemia–like in Fanconi mutated be to found been recombination has homologous repair, in BRCA2). involved as also known RAD51C, (also Recently, FANCD1 FANCN and PALB2) effectors as a known recombination (also BRIP1), as homologous the known and (also FANCJ helicase, are proteins anemia Fanconi whole second UBZ domain ( domain UBZ second whole the and domain UBZ first the of leucine and of cysteine conserved deletion the in-frame an to leading (p.Arg317_Phe387del) SLX4 317–387 of acids amino of deletion 70-amino-acid a is mutation this ( mutation negative this be for to sibling unaffected an found and heterozygous be to butane chromosomal (DEB)-induced instability ( fibr skin his ( reversion of suggestive MMC, Fig. ( (MMC) C mitomycin to sensitivity increased and ( instability genomic increased showed 1084/1 individual from (RA3083) fibroblasts and (RA3042) (LCL) line cell indiv affected ( IFAR414 and IFAR1084 tions, ( tion modifica FANCD2 normal and groups complementation anemia Registry Anemia Fanconi International ubiquitination ( complex. FANCI-FANCD2 the of downstream work Supplementary Fig. 2b Fig. Supplementary Sequencing of the complementary DNA (cDNA) from the 1084/1 1084/1 the from DNA (cDNA) complementary the of Sequencing As depletion of SLX4 in a U2OS cell line does not affect FANCD2 affect not does line cell U2OS a in SLX4 of depletion As o

1c 1a blasts from either of the affected individuals ( individuals affected the of either from blasts Fig. 1 Fig. 2 VOLUME 43 | NUMBER 2 | FEBRUARY 2011 FEBRUARY | 2 NUMBER | 43 VOLUME Division Division of Pediatric Hematology/Oncology, Herman B. Wells Center for , ). The 414/1 individual’s LCL (RA 1376) was not sensitive to to sensitive not was 1376) (RA LCL individual’s 414/1 The ). d doi:10.1038/ng.75 ( ). Fibroblasts from individual 414/1 (RA3331) but, interest (RA3331) 414/1 individual from ). Fibroblasts PALB2 c o ) and identified two families carrying germline muta germline carrying families two identified and ) blasts (RA 3331) displayed a high degree of diepoxy of degree high a displayed 3331) (RA blasts i duals are summarized in are summarized duals o Fig. 1a ubiquitination, and their products are thought to to thought are products their and ubiquitination, ), ), Supplementary Fig. 2b Fig. Supplementary 2 FANCD1 , 3 , , Arleen D Auerbach , b ), we sequenced 0 ). ). We of individual’s both this parents found 3 Department Department of Otorhinolaryngology, Heinrich Supplementary Fig. 1e Fig. Supplementary ( Fig. 2 Fig. BRCA2 1 5 Supplementary Fig. 1b Fig. Supplementary . Cells mutated in in mutated Cells . i. 1 Fig. a and and 5 ) and and ) These These authors contributed equally Table SLX4 1 d 6 ). Phenotypes of the two two the of Phenotypes ). with unassigned Fanconi Fanconi unassigned with Supplementary Fig. 2c Fig. Supplementary ). The predicted effect of of effect predicted The ). Supplementary Supplementary Fig. 2a RAD51C in the families from the 1 . The lymphoblastoid . lymphoblastoid The 4 Fig. 1 Fig.

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Supplementary Supplementary Supplementary Supplementary , f e ). have normal normal have e and and i and duals with a with duals ); however, however, ); ( Table Table BRIP1 n etics 2 2 ), ), ). ). - - - ) ) )

© 2011 Nature America, Inc. All rights reserved. of U2OS cells transfected with the indicated siRNAs and treated with 1 with treated and siRNAs indicated the with transfected cells U2OS of 1 Figure Nature Ge Nature frameshift. a 414/1 1084/1 Individual T MMC the rescued fully almost lines cell both in SLX4 wild-type of ( assays plementation com functional performed and RA3331) and (RA3083 fibroblasts intro we duced the or wildtype the mutant individuals, affected both of phenotype anemia Fanconi ( (RA3331) protein in the individual’s the full-length fibroblasts to failed identify (p.Leu672Val SLX4 against antibody the with frameshift immunoprecipitation Consequently, a to due acids amino of 671 acids amino by SLX4 followed 119 non-SLX4 N-terminal with ( Fig. c.2013+225_3147delinsCC in resulting 12 exon to 9 intron from deletion genomic large a showed Methods, Online the in described as identified 414/1, individual the in SLX4 of allele (p.Leu172Phe 22 acids by amino followed non-SLX4 SLX4 of acids amino 171 N-terminal truncated with a is protein mutation frameshift this of effect protein predicted ( father and his DNA individual of this ( product RT-PCR length full the of sequencing by (c.514delC) 2 exon in tion 2d Fig. Supplementary ( product protein shorter of a slightly presence the confirmed RA3083 line cell the from SLX4 of Immunoprecipitation (DEB)). diepoxybutane with (treatment 414/1 individual from RA3331 fibroblasts the and 1084/1 individual from lines ( symbols. half-filled by indicated are carriers Mutation symbols. filled with indicated are probands two The RB). (B, samples blood peripheral and (RA) lines cell for numbers accession showing study this in 1 with treated were or untreated left were Cells fibroblasts. RA3331 and RA3083 BJ, in shown experiment the FANCD2. ( of form non-monoubiquitinated the indicates The predictedprotein has aninternaldeletionfromaminoacids317 to387. d a able able 1 B06-0630.4 To prove that the mutations identified in in To identified mutations the prove that muta frameshift heterozygous a detected we 414/1, individual In RA3083 RA3042 F RA3321 G

ANCD2 3c siRNA APDH MMC –

e c Characteristics of Characteristics individuals with Fanconi anemia and mutations in Characterization of cell lines from individuals with Fanconi anemia with with anemia Fanconi with individuals from lines cell of Characterization The predictedprotein has 172N-terminalaminoacidsofSLX4followed by22non-SLX4aminoacidsduetoa frameshift.s.d.,standarddeviation. ). ). The predicted effect of this mutation is a truncated p.Leu672Val del4890insCC, c.2013+225_3147 p.Arg317_Phe387del c.1163+2T>A, Supplementary Fig. 3a Fig. Supplementary SLX4-1 n Fig. 2 Fig. IF + – AR1 etics Maternal allele 084 b SLX4-2 + – B06-0630.3 B06-041

lane 6). lane RA3320 RA3322 fs VOLUME 43 | NUMBER 2 | FEBRUARY 2011 FEBRUARY | 2 NUMBER | 43 VOLUME Fig. Fig. X119 a ). SLX4-3 . Error bars indicate the standard deviation (s.d.) of three replicates. ( replicates. three of (s.d.) deviation standard the indicate bars . Error + – 8.3

3 b and and a U2OS + – p.Leu172Phe c.514delC, p.Arg317_Phe387del c.1163+2T>A, RB09-1 Luc Supplementary Fig. 4 Fig. Supplementary RA3324 SLX4 ) and confirmed it in the genomic genomic the in it confirmed and ) Paternal allele 1 24.2 + – AT cDNAs into these individuals’ fs Supplementary Fig. 3b Fig. Supplementary X22) ( X22) F R RA3331 IF RA1 AR41 fs SLX4 X22 37 + – ANC 6 4 Fig. 2 Fig. c RB09-1 I were causal for the for the causal were a fs RA3323 Fig. 2 Fig. e 19 ( X119) b ) Examples of metaphases of the LCL RA3042 (no drug treatment) and fibroblast RA3083 cell cell RA3083 fibroblast and treatment) drug (no RA3042 LCL the of metaphases of ) Examples Supplementary Supplementary ) RT-quantitative PCR in U2OS cells transfected with various siRNAs against SLX4 used in used SLX4 against siRNAs various with transfected cells U2OS in PCR ) RT-quantitative a S L 1 ). The second second The ). 24.1 ). Expression Expression ). descent European American of South Indian b b b Ethnicity The predictedprotein has 671N-terminalaminoacidsofSLX4followed by119non-SLX4aminoacidsdueto a lane 5 and lane Relative mRNA i. 2 Fig. abundance

protein 0.2 0.4 0.6 0.8 1. 1.

e ). ). The 0 0 2

µ Luc ). ). M MMC for 24 h. L (long) indicates a monoubiquitinated form and S (short) S (short) and form a monoubiquitinated indicates L (long) h. 24 for M MMC - - - baseline RA3042 died at22yearsofagefromcomplications ofmetastaticdisease. tongue at21yearsofage;platelets,35,000 cells/ kidney, malformedauricleandshortstature; squamouscellcarcinomaofthe Bilateral absentthumbsandrightradial aplasia,undescendedlefttesticle,pelvic presented at9yearsofagewithisolated thrombocytopenia. Fifteen-year-old female,shortstature(height –2.1s.d.,1stpercentile);vitiligo; with with MMC treatment ( sensitivity ( blasts, showed diminished but present interaction with ERCC4/XPF ERCC4/XPF with interaction but present diminished showed blasts, p.Leu672Val diminished immun to leading experiments, multiple in lower consistently were protein Supplementary Fig. 5a ( fibroblasts RA3083 from (p.Arg317_Phe387del) and coimmun ERCC4/XPF ERCC1 the with interacted MUS81 still individuals both affected from proteins SLX4 mutant the whether tested repair therefore We crosslink in implicated previously been have MUS81, and FANCP anemia-P, that Fanconi and anemia, Fanconi of subtype new a cause biallelic that demonstrate experiments These tion. func over residual have to might which due proteins, mutant the of possibly expression assays, various the in noted we improvement slight although a cells, Fanconi individuals’ the these rescue of not phenotypes did anemia proteins mutant the of Introduction shown). not (data analysis immunofluorescence by evident as SLX4, of expression losing cells some to due likely most is breakage somal Fig. 4f ( DEB with treatment after instability chromosomal SLX4-1 SLX4 interacts with multiple factors; two of which, ERCC4/XPF ERCC4/XPF which, of two factors; multiple with interacts SLX4 SLX4 ). ). Some residual MMC sensitivity, cell cycle arrest and chromo SLX4 µ becomes an alias for for alias an becomes SLX4-2

mutations. ( mutations. M MMC for 24 h. ( h. 24 for M MMC complexes. We found that ERCC4/XPF, MUS81 and and MUS81 ERCC4/XPF, that found We complexes. Fig. Fig. 3 fs X119 mutant protein, overexpressed in RA3331 fibro RA3331 in overexpressed protein, mutant X119 SLX4-3 Phenotypic andhematologicabnormalities c a ) Protein blot analysis with a FANCD2 antibody of a FANCD2 antibody with analysis blot ) Protein and o o 0.1 a precipitated with endogenous mutant SLX4 SLX4 mutant endogenous with precipitated precipitation of the interacting factors. The SLX4 ) Protein blot analysis with a FANCD2 antibody a FANCD2 antibody with analysis blot ) Protein RA3083 µ Fig. Fig. 3 Supplementary Fig. Supplementary 4a lane 4), although the levels of the mutant SLX4 g/mL DEB d F c ) Pedigrees of the two families described described families two the of ) Pedigrees ANCD2 tub b Alpha MMC SLX4 and ulin Supplementary Fig. Supplementary . + – BJ µ l; Hb,10g/dL;MCV, 105.5fL; 0.1 RA3331 RA3083 µ + – , g/ml DEB b ), ), the late S/G2 arrest Fig. 4 Fig. s r e t t e l SLX4 Supplementary Supplementary

+ – 4c RA3331 a lane 5 and and 5 lane – mutations mutations e ) ) and the 143 S L 8 - .

© 2011 Nature America, Inc. All rights reserved. sistent with the previous findings that MUS81 interacts with the the with interacts MUS81 amino acid that 684–1,834 fragment of the SLX4 protein findings previous the with sistent ( MUS81 with not but ERCC1 and breaks includeschromatidandradialchromosomes.DEB,diepoxybutane. a Breaks permetaphase with breaks % ofmetaphases Quadriradials Triradials Chromatid breaks Total breaks Metaphases DEB concentration( with and without treatment diepoxybutane T s r e t t e l 144 S in shown are proteins exogenous the of levels Expression parallel. in analyzed were samples h Untreated later. 48 analyzed was cycle cell the and MMC, nM 100 with treated were cells Indicated treatment. MMC after ( s.d. indicate bars Error survival. percent at arrive to sample untreated the in cells of number the by divided were dose each at Total numbers counter. cell a coulter using determined was number cell the 8 days, After nM. 0–100 from ranging MMC of levels different to cDNAs SLX4 mutant the or SLX4 wildtype expressing vector the or (control) vector empty with transduced stably ( cDNA. 3 Figure understand to tools important are here described lines cell deficient SLX4- the damage, DNA of sites to SLX1, and MUS81 ERCC4/XPF, including proteins, other localize would SLX4 As substrates. nated ubiquiti to K63 binding through of damage sites to the may localize ( alone GST with seen levels background to reduced was binding the each UBZ domain were mutated to alanines ( ( of ubiquitin showed binding of the isolated UBZ domains of SLX4 to the K63 chains We therefore performed (ref. RAP80 of domains UBZ tandem the for DNA shown as of damage, sites the at proteins repair of chains ubiquitin to complex SLX4 the of binding the disrupt might 1084/1 individual from SLX4 mutant the in domains UBZ tandem of the absence the that thesized ( MUS81 showed greatly diminished interaction with ERCC4/XPF, ERCC1 and cells RA3331 from SLX4 of terminus N the recognizing antibody an in the p.Leu672Val BJ foreskinfibroblastsfromahealthydonorwereusedasnormalcontrol. Fig. 4 Fig. able able 2 upplementary Figure 4a–c Figure upplementary As As UBZ are domains ubiquitin with to known interact a

c a 100

Survival (% of untreated) , compare lane 7 and 9), suggesting the possibility that SLX4 SLX4 that possibility the suggesting 9), and 7 lane compare , ) Complementation of MMC sensitivity. We exposed fibroblasts fibroblasts We exposed sensitivity. MMC of ) Complementation 20 40 60 80 Chromosome Chromosome breakage analysis in the indicated cell lines Complementation of RA3083 and RA3331 cells with the the with cells RA3331 and RA3083 of Complementation Supplementary Fig. 5b Fig. Supplementary 0 b 2 0 Fig. 4 Fig. All cellshTERT 4 0

MMC (nM) µ 6 0 g/ml) fs c lane 8). When the two conserved cysteines from cysteines lane 8). When the two conserved X119 X119 mutant protein. Immunoprecipitation with p.Arg317_Phe387del RA3083 RA3083 +WTSLX4 RA3083 +control BJ +control 8 0 b 0.73 ) Complementation of the cell cycle defect defect cycle cell the of ) Complementation in vitro 0 30 29 41 56 RA3042 . Quantification of the data is shown in in shown is data the of . Quantification 0 1 5 (LCL) 100 + 123 221 7.6 0.1 90 44 29 ubiquitin binding assays ( 5

lane 6). lane 0.15 Fig. 4 Fig. RA3083 13 53 E6E7 0 1 6 8 0 b Survival (% of untreated) 140 100 4.4 0.1 60 80 20 40 81 16 92 32 lane 3). This result is con is result This 3). lane 0 8

Supplementary Supplementary Fig. 2c 2 0 0.14 RA3331 14 50 All cellsE6E7 4 0 E6E7 0 0 7 7 0 MMC (nM 7 6 0 , , which is deleted 100 123 217 7.0 0.1 11 36 31 p.Leu672Val RA3331 RA3331 +WTSLX4 RA3331 +control 8 0

b Total numberof ) 1 Fig. 4 7 0 0.11 , , we hypo 100 11 63 E6E7 + 0 0 1 6 8 S SLX4 BJ c upplementary Figure 4d,e Figure upplementary fs ) ) that 0.16

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b the interacting proteins and the predicted protein effect of of effect protein predicted the and proteins interacting the ( 2 Figure onset of childhood solid tumors and acute early very have recombination, homologous for essential are which in mutations with anemia Fanconi with clue. Individuals an provide important also individuals affected of the pathway. Phenotypes anemia Fanconi the in function (FANCP) SLX4 of the importance the define to DNA ultimately and of SLX4 for interactions are which the repair of essential cross-linked WT, 9). 7 to wildtype. (lanes blotting protein straight in SLX4 identify not does antibody the that Note bands. crossreacting the indicate Asterisks antibody. SLX4 the or IgG) (control antibody rabbit a control using immunoprecipitation to fibroblasts RA3331 and RA3083 BJ, primary of extracts cell We subjected lines. cell the in protein SLX4 mutant the ( individuals. IFAR414/1 and IFAR1084/1 in mutations a

Cell count Cell count IFAR1084/ b a ) Schematic of SLX4 (based on ref. 7) showing the domain architecture, architecture, domain the showing 7) ref. on (based SLX4 of ) Schematic IFAR414/ 1,000 2,000 3,000 1,000 2,000 3,000 4,000 MMC WT SLX4 MM – C 0 0 SLX4 VOLUME 43 | NUMBER 2 | FEBRUARY 2011 FEBRUARY | 2 NUMBER | 43 VOLUME

IP: SLX4 1 1 +vector control + – BJ hTERT 1 BJ E6E7

2 1 BJ

27% 17% Control IgG 172 is defective in two individuals with Fanconi anemia. anemia. Fanconi with individuals two in defective is UBZ UBZ 292 ∆ . . WT, wildtype. 1 1 1 C20orf94 317-387 st – +

RA3083 allele:p.Leu172Phe 323 UBZ 26% UBZ 2 33% 333 2 MSH2 4 3 RA3331 361 MSH3 RA3083 hTERT +vector control +vector control RA3331 E6E7 XPF + – BJ ERCC1 SLX4 Ab

23% 14% PALB2

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RA3331 SLX1 SBD SBD 1,742 + p.Arg317_Phe387del BRCA2 * * + p.Leu672Val fs Nature Ge Nature X119 b RA3083 hTERT p.Arg317_Phe387del RA3331 E6E7 1,834 ) Analysis of ) Analysis + – SLX4 Both alleles:

27% 13% ( FANCD1

+ fs n X119 51% 56%

etics 19,20 ), ), . © 2011 Nature America, Inc. All rights reserved. subjected cell extracts of primary fibroblasts (BJ, RA3083 and RA3331) to immunoprecipitation using the SLX4 antibody. We identified interacting interacting We antibody. identified SLX4 the using immunoprecipitation to RA3331) and RA3083 (BJ, fibroblasts primary of extracts cell subjected 4 Figure Nature Ge Nature for communicating unpublished results. H.H. is supported by the Deutsche Boston, Massachusetts, USA for reagents, E. Foley for advice and J. de Winter participation in this study. We thank the Harper Lab, Harvard Medical School, We are grateful to the affected individuals and their families for their Note: Supplementary information is available on the and NM_032444.2 NP_115820.2. sequences: reference following the with NCBI codes. Accession online the at http://www.nature.com/naturegenetics/. paper the of in version available are references associated any and Methods Met identified. be could mutations ing predispos other no where pedigrees from individuals in sequenced cancers ovarian develop and of breast risk familial high ing a with associated are disorder, anemia–like ( ( genes anemia Fanconi three in tions muta germ-line Because the cells. of human in function pathway anemia the Fanconi into insight provide will organism model this in work the and SLX1, and ERCC4/XPF of orthologs with interacts SLX4, Yeast human like Slx4, components. pathway anemia Fanconi other any have not do which eukaryotes, lower in conserved is that SLX4. with interact that MUS81, and ERCC4/XPF nucleases, other the on depends repair the that and repair crosslink junction of function SLX4 be the in essential the during somatic compartment Holliday the that step an of integral hom resolution, suggests This components. complex in seen that FANCI-FANCD2 the or core to anemia Fanconi the in mutations akin with more phenotype milder a show having anemia Fanconi with Individuals MUT,WT, type; mutated. wild proteins. GST the of staining Ponceau shows panel bottom The antibody. ubiquitin with immunoblotted a PAGEand on gel separated beads, GST to binding by purified ubiquitin, of forms indicated the with p.Cys339Ala) and p.Cys336Ala p.Cys299Ala, p.Cys296Ala, 251–402 acids amino (SLX4 alanines to mutated cysteines four with domains UBZ ( 30 of presence the in antibody HA or antibody p.Leu672Val the or SLX4 wild-type vector, control HA-tagged expressing cells E6E7 RA3331 ( antibodies. indicated the with immunoblotting by proteins Ac c ERCC4/ FANCN a ERCC1 MUS81 ) Interaction of the UBZ domains with ubiquitin. We incubated GST-purified GST control, wild-type UBZ domains (SLX4 amino acids 251–402) and and 251–402) acids amino (SLX4 domains UBZ wild-type control, GST GST-purified We incubated ubiquitin. with domains UBZ the of ) Interaction SLX4 (FANCP) represents a second protein (besides FANCM) FANCM) (besides protein second a represents (FANCP) SLX4 k SLX4 XPF now h o

Interaction of mutant forms of SLX4 with its partners and with ubiquitin. ( ubiquitin. with and partners its with SLX4 of forms mutant of Interaction ) ) and ds IP: l e n d 2 1 etics gment

BJ Control BRIP1 IgG RA3083 The The

4 3 VOLUME 43 | NUMBER 2 | FEBRUARY 2011 FEBRUARY | 2 NUMBER | 43 VOLUME s ( RA3331 FANCJ (C terminus) SLX4 BJ SLX4 Ab 6 5 RA3083 reference sequences are deposited in in deposited are sequences reference )) )) and

RA3331 8 7 o BJ logous recombination, might not might recombination, logous RAD51C input 2% RA3083 BRCA1 21–24 9

SLX4 RA3331 N µ a g of HA peptide. We identified interacting proteins by immunoblotting with the indicated antibodies. antibodies. indicated the with immunoblotting by proteins interacting We identified peptide. HA g of t , , mutated in an Fanconi u , , r ERCC4/ b ERCC1 e MUS81 SLX4 (

( G FANCP XPF e FANCD1 IP: HA n e t should also be be also should i c s b website. ) Analysis of SLX4-interacting partners in RA3331 cells. We subjected cell extracts of extracts cell We subjected cells. RA3331 in partners SLX4-interacting of ) Analysis

) mutations mutations ) individuals individuals 2 1 HA Ab Control ) , PALB2 ,

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6 5 WT SLX4 fs 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprintsandpermissions at online available is information permissions and Reprints Published online at http://www.nature.com/naturegenetics/. The authors declare no competing interests.financial R.D. The manuscript was written by A.S. with help from other authors. Mutation analysis and studies functional were performed by A.S., Y.K., F.P.L. and to Fanconi anemia subgroups was performed by A.S., F.P.L., H.H. and A.D.A. was collection done by A.D.A., F.P.L. and A.S. Characterization with respect The study was bydesigned A.S., Y.K. and F.P.L. Subject recruitment and sample and is a Rita Allen Foundation and an Irma T. Hirschl Scholar. supported by the Burroughs Wellcome Fund Career Award for Medical Scientists Forschung network for Bone Marrow failure Syndrome, and FoneFA. A.S. is Forschungsgemeinschaft SPP1230, the Bundesministerium für Bildung und COM AU X119

p.Leu672Val

T rt, K. Kratz, A. Smogorzewska, I. Garcia-Higuera, relationships functional and Structural S.C. West, & N. McDonald, A., Ciccia, J.M. Svendsen, I.M. Munoz, S. Fekairi, the P.Knipscheer, guards pathway anemia fanconi the How A.D. D’Andrea, & G.L. Moldovan, Fanconi of Susceptibility S.R. Wolman, & diagnosis. A.D. Auerbach, its and anemia Fanconi A.D. Auerbach, sensitizes cells to interstrand crosslinking agents. crosslinking interstrand to cells sensitizes repair. DNA for required paralog FANCD2 pathway. common a proteins. of family (2008). XPF/MUS81 the of repair. DNA for required is and resolvase repair. DNA for required is BTBD12 endonucleases. repair/recombination DNA multiple repair. cross-link interstrand DNA genome. carcinogens. by damage chromosome (2009). 7 H

P Control a O ET input ) Analysis of SLX4-interacting partners in SLX4 mutant cells. We cells. mutant SLX4 in partners SLX4-interacting of ) Analysis 1% 8 R R WT SLX4

I fs CONT NG X119 9 Annu. Rev. Genet. Rev. Annu. p.Leu672Val et al. et t al. et FI t al. et fs N et al. et RIBU X119 SLX4 alteration to immunoprecipitation using HA using immunoprecipitation to alteration SLX4 X119 MUT WT Human SLX4 is a Holliday junction resolvase subunit that binds that subunit resolvase junction Holliday a is SLX4 Human A t al. et Deficiency of FANCD2-associated nuclease KIAA1018/FAN1 nuclease FANCD2-associated of Deficiency NC

et al. et fs Coordination of structure-specific nucleases by human SLX4/ by human nucleases structure-specific of Coordination et al. et X119 / The Fanconi anemia pathway promotes replication-dependent promotes pathway anemia Fanconi The Mol. Cell Mol. . T IAL I 100 150 c amla BB1/L4 sebe a oldy junction Holliday a assembles BTBD12/SLX4 Mammalian I 25 37 50 10 15 20 25 37 50 75 Interaction of the Fanconi anemia proteins and BRCA1 in BRCA1 and proteins anemia Fanconi the of Interaction ON Identification of the FANCI protein, a monoubiquitinated a protein, FANCI the of Identification

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Hofmann, K. Ubiquitin-binding domains and their role in the DNA damage response. Kutler, D.I. Vaz,F. A. Smogorzewska, FANCI- with MacKay, acts C. FAN1 J. Huang, & J. Chen, J., Yuan, G., Ghosal, T., Liu, DNA Repair (Amst.) Repair DNA (IFAR). Genet. (2010). 36–47 repair. crosslink interstrand DNA for necessary nuclease associated FANCD2. monoubiquitinated by damage DNA to repair. (2010). cross-link interstrand DNA promote to FANCD2

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101 A 20-year perspective on the International Fanconi Anemia Registry Identification of KIAA1018/FAN1, a DNA repair nuclease recruited , 1249–1256 (2003). 1249–1256 , t al. et

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© 2011 Nature America, Inc. All rights reserved. using the Invitrogen SuperScript III Reverse Transcriptase kit. SLX4 RT-PCRSLX4 kit. Transcriptase Reverse III SuperScript Invitrogen the using synthesized then were cDNAs First-strand Kit. Mini Plus RNeasy QIAGEN cDNA sequencing. and genomic proteins. E7 and HPVE6 using transformed were or fibroblasts were using a immortalized catalytic subunit of telomerase (hTERT) from ATCC. and were Subjects’ obtained fibroblasts foreskin are normal cells BJ oxygen. 3% in grown were Fibroblasts acids. amino non-essential and FBS were above 20% with Fibroblasts FBS. grown except as above 15% with except as supplemented (RPMI) medium Institute Park Memorial Roswell in grown 0.1 mg streptomycin per ml from Lymphoblast (all Invitrogen). were lines cell (DMEM) supplemented with 10% (v/v) FBS, 100 units of per ml penicillin and Culture. Cell New York, New York, studies. the approved USA University, The Rockefeller The consent. of Board written Review informed Institutional obtaining after Registry Anemia Fanconi with Fanconi anemia and their family members registered in the International Subjects. O doi:10.1038/ng.750 of SLX4 (Bethyl). 251–402 acids against amino raised an antibody Ab-2), Mus81 (Abcam, MTA30 2G10/3), ubiquitin (Millipore, MAB1510) and 269A and A302-270A), ERCC4/XPF (Neomarkers Ab-1), ERCC1 (NeomarkersA302- (Bethyl SLX4 MMS-101R), (Covance HA 11814460001), (Roche GFP Antibodies. pMSCVneo lines. cell BJ and RA3083 into immortalize to subcloned were pWZLhTERT USA) cells. the primary to and used transform (Clontech) Massachusetts, Boston, Medical School, Harvard Lab, Howley from (gift genes E6E7 HPV16 clonase LR (Invitrogen). using pDEST15 or vectors pHAGE into recombined were tives (ref. pDONR223 into recombined and amplified were alleles Mutant USA. Massachusetts, Boston, School, Medical Harvard Plasmids. amplify. not did allele wildtype the because homozygous appears ( cDNA the in observed 3e Fig. the subject’s and the mother’s DNA but not the father’s DNA ( conditions we used, but the deleted allele resulted in a 1,931-bp amplicon from PCR the under to amplify failed bp) (6,821 amplicon wild-type resulting The deletion. the flanking primers using up set was assay PCR genomic a result, ( c.2013+225_3147del4890insCC of deletion with c.2013+224, in of and its nucleotides hence two insertion place: cytosine c.2013+225_3147 through IVS9 of retention revealed analysis rs714181). Amplification of parts of the cDNA(p.Pro1122Leu, followed by a detailed sequence c.3365C>T and rs76488917) (p.Ser1054Ser, exon c.3162G>A in 12: markers polymorphic informative on based likely be to found was deletion genomic A DNA. maternal the and subject’s the on done was exons coding the of sequencing genomic full line, cell RA3331 the in allele second IFAR414/1. in allele second the of Identification sequencing. for suitable also were primers PCR the using the USB Exo-Sapit kit set up before In being for most cases, sequencing. in shown ers Table 1 639, 640 and 641 were SLX4 used to ( the sequence full-length New (South Plainfield, Jersey, USA). Primers 623, 627, 628, 629, 630, 633, 634, Inc. Genewiz, by done sequencing, for up set being before kit Exo-Sapit USB the using up cleaned were products PCR kit. DNAPolymerase Pfx Platinum was done using primers 621 and 624 ( NLIN ). Direct sequencing of the mutant amplicon confirmed the result result the confirmed amplicon mutant the of sequencing Direct ). ). Genomic PCR was ). done QIAGENGenomic using Taq prim with Polymerase E E Cell lines and Cell genomic DNA samples were from derived individuals The wild-type SLX4 cDNA was a kind gift from the Harper Lab, Lab, Harper the from gift kind a was cDNA SLX4 wild-type The M The following antibodies were used: FANCD2 (Novus NB100-182), U2OS cells were grown in Dulbecco Modified Eagle medium medium Eagle Modified Dulbecco in grown were cells U2OS ET Supplementary Table 1 Table Supplementary H O DS upeetr Fg 3c Fig. Supplementary RNA from the using cell lines was extracted Supplementary Fig. 2d Fig. Supplementary Supplementary TableSupplementary 1 . The PCR products were cleaned up up cleaned were products PCR The . ). The sequencing result result sequencing The ). 25). pDONR223 deriva pDONR223 25). In order to identify the the identify to order In ). To confirm this this confirm To ). ) ) and Invitrogen Supplementary Supplementary Supplementary Supplementary 2 6 was used was used - - (FACS), cells were spun down, resuspended in propidium iodine mix (1 ml ml (1 mix iodine sorting propidium in cell resuspended down, spun were cells (FACS), fluorescence-activated before minutes at Thirty °C −20 at overnight. stored least was suspension the and dropwise, added were ethanol were cells 300 in Collected h. resuspended 48 for grown were and MMC nM 100 with treated studies. cycle Cell MAB1510). (Millipore, antibody ubiquitin with noblotted immu and on a Bis-Tris (Invitrogen) gel loaded and boiled were samples the buffer, binding with times five washing After °C. 4 at h 3 for incubated and 200 and NEM mM 2 Triton, 0.1% glycerol, 10% NaCl, mM 150 100 in Biochem) (Boston Ub-K63 or alone as a control were added to 7.5 or GST mutants, ZNF2 and ZNF1 251–402, acids amino SLX4 GST 251–402, Ubiquitin binding assay. control. the as used was Actin facturer’s instructions. super mix Platinum (Invitrogen) cybergreen was used according to the manu PCR. quantitative RT centration of 50 nM. siRNAs (Invitrogen) are shown in Lipofectamine RNAiMAX as suggested byRNAi. the manufacturer with a final siRNA con in shown primers using (Agilent) Mutagenesis. 26. 25. survival. percent at the arrive to sample untreated the in number cell the to normalized was treatment MMC after number cell The Coulter). (Beckman Counter Coulter Z2 a using determined were numbers from nM. 8 at was 0–100 After added concentrations days final in cell culture, 10 × 2.5 of density a at licate assay. sensitivity C Mitomycin (Invitrogen). gel Tris-Acetate 3%–8% Novex on fractionated size and buffer SDS sample in buffer, lysis were eluted in the tris-glycine immunoprecipitates 10 and two or One milligrams of protein (Calbiochem). extract was incubated with 5 inhibitors phosphatase and (Roche) Inhibitors protease with supplemented NP-40) 0.5% and NaCl mM 150 Tris, mM (50 Immunoprecipitations. application. Metafer Metasystems the using scanned and min 60 for °C 40 at dried fully were distilled slides by the water, followed Buffer Gurr fresh with rinsing After min. 3 for Buffer at 40 °C for 60 min before staining with Giemsa KaryoMAX (Invitrogen) Gurr dried air and slides wet onto dropped needed, when and, °C 4 at stored were Cells vol/vol). (3:1 acid acidic methanol:glacial prepared freshly the in fixed and KCl M 0.075 in °C 37 at min 10 for incubated harvested, fibroblasts, for (0.17 colcemid with arrested h, analysis. Breakage software FlowJo the using Inc). Star, (Tree performed was analysis cycle (Becton Cell FACSCalibur using Dickinson). analyzed and mg/ml)) 1 of solution stock (of 10 PBS,

Dolma, S., Lessnick, S.L., Hahn, W.C. & Stockwell, B.R. Identification of genotype- W.C. of Hahn, Identification S.L., B.R. Lessnick, Stockwell, S., & Dolma, P. Lamesch, human tumor cells. tumor human engineered in screening chemical lethal synthetic using agents antitumor selective genes. human 10,000 over representing hr itreig N (iN) rnfcin wr promd using performed were transfections (siRNA) RNA interfering Short µ µ l of Protein A/G PLUS-Agarose (Santa Cruz). Following five washes washes five Following Cruz). (Santa PLUS-Agarose A/G Protein of l l RNase (of stock solution of 20 mg/ml), 10 ml propidium iodine iodine propidium ml 10 mg/ml), 20 of solution stock (of RNase l Mutagenesis was performed using a multisite mutagenesis kit kit mutagenesis multisite a using performed was Mutagenesis t al. et Cells were treated with 0.1 with were treated Cells For cell cycle analysis, cells were left untreated or were were or untreated left were cells analysis, cycle cell For OFoe 31 a eore f ua oe raig frames reading open human of resource a v3.1: hORFeome µ Cancer Cell Cancer l PBS. While vortexing, 700 700 vortexing, While PBS. l Superscript III reverse transcriptase followed by by followed transcriptase reverse III Superscript For immunoprecipitations, cells were lysed in MCLB Five micrograms of purified GST SLX4_amino acids 4 cells per well. Twenty-four hours later, MMC MMC later, hours Twenty-four well. per cells

µ Cells were plated in a six-well plate in trip in plate six-well a in plated were Cells 3 g per ml of media) for 20 min (LCL) or 2 h 2 or (LCL) min 20 for media) of ml per g , 285–296 (2003). 285–296 , Supplementary Table 2 Supplementary µ l l GST and beads 1 µ l binding buffer (50 mM Tris, pH 7.5, 7.5, Tris,pH mM (50 buffer binding l Genomics µ g g DEB ml for of per 72 media

µ 89 g g of the indicated antibody µ Supplementary Table 3 , 307–315 (2007). 307–315 , l of ice cold 100% (v/v) (v/v) 100% cold ice of l µ Nature Ge Nature g g mono-Ub, Ub-K48 . µ g/ml BSA) BSA) g/ml n etics . - - - - Mutations of the SLX4 gene in Fanconi anemia. Y. Kim, F. P. Lach, R. Desetty, H. Hanenberg, A. D. Auerbach, A. Smogorzewska Supplementary Figures and Tables

Figure S1. Damage sensitivity of patient cell lines. A. Mitomycin C (MMC) sensitivity of lymphoblasts from IFAR 1084 family. B. MMC sensitivity of lymphoblasts from IFAR 414 family. C. and D. UV sensitivity of the indicated cell lines. E and F. Camptothecin (CPT) sensitivity of the indicated cell lines. Cells were treated in triplicate with different levels of indicated damaging agents. After 8 days, cell number was determined using a coulter counter. Total cell numbers at each dose were divided by the number of cells in the untreated sample to arrive at percent survival. Error bars indicate standard deviation.

RA3042 RA3321 RA1376 BJ + CONTROL BJ + CONTROL a RA3320 RA3322 b c d 100 100 RA3323 RA3083+ CONTROL RA3331+ CONTROL 90 90 RA3083 + WT SLX4 RA3331 + WT SLX4 RA3083 + p.R317_F387del RA3331+ p.L672VfsX119 80 80 100 100 all hTERT all E6E7 70 70 60 60 80 80 50 50 60 60 40 40 30 30 40 40

survival (% of untreated) 20 survival (% of untreated) 20 survival (% of untreated) 10 10 survival (% of untreated) 20 20 0 0 0 20 40 60 80 100 0 20 40 60 80 100 0 0 MMC (nM) MMC (nM) 0 10 20 30 40 50 60 0 10 20 30 40 50 60 2 UV (J/m2 ) UV (J/m ) BJ + CONTROL BJ + CONTROL e RA3083+ CONTROL f RA3331+ CONTROL RA3083 + WT SLX4 RA3331 + WT SLX4 RA3083 + p.R317_F387del RA3331+ p.L672VfsX119 100 100 all hTERT all E6E7

80 80

60 60

40 40 survival (% of untreated) 20 survival (% of untreated) 20

Nature Genetics: doi:10.1038/ng.750 0 0 0 2 4 6 8 10121416 0 2 4 6 8 10 12 14 16 CPT (nM) CPT (nM) Figure S2. Sequencing of SLX4 in the IFAR1084. A. Chromatogram displaying skipping of the exon 5 as identified by direct sequencing of full length RT-PCR product from RA3042 cell line. B. Chromatogram displaying the splice donor site mutation and its segregation in the genomes of the IFAR1084 family. Genomic DNA from the fibroblast RA3083 cell line derived from the proband and the genomic DNA isolated from the peripheral blood (PB) of the other individuals were amplified and sequenced as described in Online Methods. C. Alignment of the UBZ domains of SLX4 from Mus musculus (Mus m.), Rattus norvegicus (Rattus.n), Homo sapiens (Homo s.), Pan troglodytes (Pan t.), Bos Taurus (Bos t.), Gallus gallus (Gallus g.), Danio Rerio (Danio r.). The residues that coordinate Zn are high- lighted in red. Grey highlights the residues that are deleted in the patient from IFAR 1084 family. Magenta stars point to the Cys residues mutated for the experiment shown in Figure 4C (UBZ MUT). D. Cell extracts of primary BJ and RA3083 cells were subjected to immunoprecipitation using the SLX4 antibody and immunoprecipitates were bloted with another SLX4 antibody.

RA3042 proband cDNA a EXON 5 sequence EXON 6 sequence WT RA3042

RA3042

EXON 4 sequence EXON 6 sequence

IFAR1084 b c Mus m. ALQQEFRREE--ASSHHDSLEEKGLFFCQMCQKNLSAMNVTRREQHVNRCLDEAEKAQR- 151 Rattus n. ALQQEFRKEE--ASSRPDSLEEKGLFFCQICQKNLSAMNVTRREQHVNRCLDEAEKALR- 161 c.1163+2T>A Homo s. TLQQEFARVG--ASAHDDSLEEKGLFFCQICQKNLSAMNVTRREQHVNRCLDEAEKTLR- 327 Pan t. TLQQEFARVG--ASAHDDSLEEKGLFFCQICQKNLSAMNVTRREQHVNRCLDEAEKTLR- 327 Bos t. ALQQELGQE--QASTPDDSLEETGLFFCQICQKNLSAMNVTRRQQHVNRCLDEAEKALS- 337 EXON 5 IVS5 Gallus g. ALQQETQKES------LESLEDAGLFFCQICQKDLSAMNSTRREQHVNRCLDEMEEAQMS 196 Danio r. RLQQELDREAR---AAAGDVEDGGLFFCQLCQKDLSSMSPSLRTQHINRCLDASESSAPS 194 ref. seq *** .:*: ******:*:*:**:*. : * **:***** *.:

Mus m. -----PASPRIPDCPICGKPFLTTKSRISHLKQCAVRMEVGPQLLLQAVRLQTAQPEVDG** 206 * Rattus n. -----PAPPQIPDCPICGKQFLTAKSRISHLKRCAVRMEVGPQLLLQAVRLQTAQPEAGS 216 WT Homo s. -----PSVPQIPECPICGKPFLTLKSRTSHLKQCAVKMEVGPQLLLQAVRLQTAQPEGSS 382 Pan t. -----PSVSQIPECPICGKPFLTLKSRTSHLKQCAVKMEVGPQLLLQAVRLQTAQPEGSS 382 Bos t. -----FPTPLIPECPICGKPFLTLKSRISHLKQCAVKMEVGPQLLLQAVRLQGAQPAGAS 392 Gallus g. S----SSKPAVPECPICGKQFQTPQSRVSHLKRCAVEMDVPPKMLLQAVQMQVSTLG--- 249 Danio r. TSHHSQPRPRVPECPICGKSFKSEKSRSAHLKRCSSDMGVKPNDLLQALRRQAAETE--S 252 PATIENT . . :*:****** * : :** :***:*: * * *: ****:: * : (RA3083) ** control SLX4 BROTHER d IP: IgG Ab

FATHER BJ RA3083BJ RA3083 MOTHER WT anti-SLX4 MUT Nature Genetics: doi:10.1038/ng.750 Figure S3. Sequencing of SLX4 in the IFAR414. A. Chromatogram displaying heterozygosity for the c.514delC mutation in direct sequencing of the cDNA from RA1376 cell line. B. Chromatogram displaying the first heterozygous mutation in the genomic DNA from the fibroblast RA3331 cell line derived from the proband and the genomic DNA isolated from the PB of the other individuals. C. Chromatogram displaying the second heterozygous mutation in IFAR414 family. Genomic DNA from the fibroblast RA3331 and RA1376 cell lines derived from the proband and the genomic DNA isolated from the mother‘s peripheral blood were amplified and sequenced as described in Online Methods. Note that there is apparent homozygosity in the patient and the carrier due to sequencing of a PCR product detecting only the allele carrying a genomic deletion. D. Chromatogram displaying heterozygosity for the deletion/insertion mutation in RA1376. The 3’ junction is shown on the left and the 5’ junction is shown on the right. E. PCR to identify the mutant deletion/insertion in the SLX4 allele in the indicated cell line and peripheral blood samples. Father’s blood (sample RB09-1124.1), Mother’s blood (sample RB09-1124.2).

RA1376 direct cDNA sequencing-c.514delC IFAR414 mut1 a b c.514delC WT ref. seq * C T TT C C G AA G A G AAAA CC GGGAAA c.514delC RA1376 C C T TT C C G AA G A G AAAA CC GGGAAA WT MOTHER

IFAR414 mut2 c c.2013+225_3147del4890insCC PATIENT C T TT C C G AA G A G AAAA CC GGGAAA c.2013+224C C Tc.3148TT C C G AA G A G AAAA CC GGGAAA (RA3331) CT TTC C G AA G AG C T TTC CC G AA GAG IVS9 EXON12

FATHER PATIENT CT TTC C G AA G AG C T TTC CC G AA GAG (RA3331)

PATIENT d RA1376 direct cDNA sequencing RA1376 direct cDNA sequencing (RA1376) c.2013+225_3147del4890insCC c.2013+225_3147del4890insCC EXON 9 EXON10 EXON12 WT WT MOTHER

CC *insertion* e

RA1376 CC C C CTTT CGGG C EXON10 EXON12 CCCGGGG T T C AT probandfather’s (RA1376)mother’s blood blood water G T AA G AA C T GG C T IVS9 IVS9 G A C TT T G CCCC C

EXON 9 IVS9/EXON10 IVS9/EXON12 EXON12 heterozygous heterozygous 2 kb CC *insertion* 1.6 kb

Nature Genetics: doi:10.1038/ng.750 Figure S4. Complementation of RA3083 and RA3331 cells with SLX4 cDNAs. A. Expression of SLX4 alleles in RA3083 hTERT cells used in studies of MMC sensitivity and cell cycle analysis after MMC treatment. WT, mutant SLX4 (MUT) and a degradation (DEG) product are indicated. B. Expression of SLX4 alleles in RA3331 E6E7 cells used in MMC sensitivity, breakage and cell cycle studies. C. Expression of SLX4 alleles in RA3083 E6E7 cells used in breakage studies. D. Quantification of cell cycle data shown in Figure 3B top panel. The experiment was done in triplicate and error bars indicate standard deviation. E. Quantification of cell cycle data shown in Figure 3B bottom panel. The experiment was done in triplicate and error bars indicate standard deviation F. Complementation of the chromosome breakage phenotype. Indicated cells were treated with DEB at 0.1 μg/ml and chromosomal abnormalities were analyzed on metaphase spreads.. a b c SLX4 SLX4 +vector control +vector control +WT SLX4 +WT +p.L672VfsX119

WT SLX4 +vector control +vector control +WT SLX4 +WT +p.R317_F387del +WT SLX4 +WT +p.R317_F387del WT +vector control +vector control anti-HA MUT DEG anti-HA anti-HA

anti-GAPDH MUT anti-alpha tubulin

RA3083 hTERT anti-GAPDH RA3083 E6E7 BJ hTERT BJ E6E7 RA3331 E6E7 BJ E6E7

RA3083 E6E7 RA3331 E6E7 d e f p.R317_ p.L672V F387del fsX119 - MMC +MMC CONTROL WT SLX4 CONTROL WT SLX4 - MMC +MMC SLX4 SLX4 60 70 DEB concentration 0.1 0.1 0.1 0.1 0.1 0.1 50 60 µg/ml

40 50 # Metaphases 43 53 33 32 50 57 40 30 # Total Breaks 156 49 78 401 175 306 30 20 20 # Chromatid breaks 100 35 46 311 169 232 10 10 % of cells in late S/G2 stage % of cells in late S/G2 stage # Triradial 20 6* 9 34 2 31 0 0 RA3083 hTERT RA3331 E6E7 # Quadriradial 8 1* 7 11 1 6 % Metaphases BJ E6E7 51 13 48 97 22 74 BJ hTERT WT SLX4 WT SLX4 with breaks vector control SLX4 vector control SLX4 p.L672VfsX119 # Breaks Nature Genetics: doi:10.1038/ng.750p.R317_F387del per metaphase 3.6 0.9 2.4 12.5 3.5 5.4 *1 cell had 3 triradial and one quadriradial chromosomes Figure S5. Analysis of SLX4 interacting partners in SLX4 mutant cells. A. Cell extracts of primary BJ and RA3083 cells were subjected to immunoprecipitation using the SLX4 antibody. Interacting proteins were identified by immunoblotting with the indicated antibodies. Asterisk indicates SLX4 proteins. B. BJ E6E7 and RA3331 E6E7 cells were subjected to immunoprecipitation using antibody #392, which recognizes the N-terminus of SLX4. Interacting proteins were identified by immunoblotting with the indicated antibodies.

a control SLX4 SLX4 5% IP: IgG Ab +peptide input

BJ RA3083BJ RA3083BJ RA3083BJ RA3083 anti-SLX4 * *

anti-ERCC4/XPF

anti-MUS81

anti-ERCC1

anti-ERCC1 lower exposure 1 2 3 4 5 6 7 8

b control SLX4 1% IP: IgG Ab #392 input BJ RA3083 RA3331 BJ RA3083 RA3331 BJ RA3083 RA3331

anti-SLX4

anti-XPF

anti-MUS81

Nature Genetics: doi:10.1038/ng.750anti-ERCC1 1 2 3 4 5 6 7 8 9 Supplementary Table 1. Cloning and sequencing primers PCR Exon/ Product Intron (i) Primer Name Forward Primer Primer Name Reverse Primer (bp) 2 FL043 BTBD12-SLX4 ex2-1F CCTCCCTGGAAGAACTGGCATAAA FL044 BTBD12-SLX4 ex2-1R TCTTCAGAGGAGCGAGGGTCAATC 768 2 FL045 BTBD12-SLX4 ex2-2F AGGCTTCTACTTGGGTTCACTTTC FL046 BTBD12-SLX4 ex2-2R GCTGGCTGCCTGTTTTTGTAA 711 3 FL019 BTBD12-SLX4 ex3F ACCACCACCACTACCTAACG FL020 BTBD12-SLX4 ex3R GACTCTTCATTCTCTGGCTGGAT 675 4 FL005 BTBD12-SLX4 ex4F GTGGAGCTGGGTTCTGGTTTCTTT FL006 BTBD12-SLX4 ex4R TCACGGCAGCTCTATCCACAATCC 934 5 FL007 BTBD12-SLX4 ex5F GGCGGACATTTGGAGCATTTCTAC FL008 BTBD12-SLX4 ex5R TGGCTCACTGCAACCTCTACCTTC 889 i5 FL009 BTBD12-SLX5 ivs5-1F TGGACGCAGTAGTTCACACCT FL010 BTBD12-SLX5 ivs5-1R AGCAGTAGTCCCCAACCTTTTT 870 i5 FL011 BTBD12-SLX5 ivs5-2F AAACCATTCCCCTACTCCTGTC FL012 BTBD12-SLX5 ivs5-2R CACCCCTGCCATGCTAAATAC 939 i5 FL013 BTBD12-SLX5 ivs5-3F AAGGCGATAGCTGTTTGTCC FL014 BTBD12-SLX5 ivs5-3R GGGTCTGGGTTCCTTCACG 1105 6_7 FL015 BTBD12-SLX5 ex6_7F TCGGAAGTATTGATGGGTGAAG FL016 BTBD12-SLX5 ex6_7R GTAGTTGCTGGACAGAGAAGAAAG 1171 i7 FL081 BTBD12-SLX4 ivs7F CTCAGGCACATGGATAGGCTTTTG FL082 BTBD12-SLX4 ivs7R CCACGTGGTCACTGTCATTG 946 8_9 FL021 BTBD12-SLX4 ex8_9F TTGGCCGTGACAGAGGAGGAGCAT FL022 BTBD12-SLX4 ex8_9R CCAGAGGCCAGCGGTGAGC 1099 i9 FL053 BTBD12-SLX4 ivs9-1F CCGGCGGCCTTCCTCTGACTG FL054 BTBD12-SLX4 ivs9-1R GCTGCGGGATACCTGGGCTTTGA 763 i9 FL055 BTBD12-SLX4 ivs9-2F AGCGCGGTTGCCCCATCCA FL056 BTBD12-SLX4 ivs9-2R GGCGCCAAAGTCAGCAACCAG 722 10 FL023 BTBD12-SLX4 ex10F CATCGTGGGAAGCGTTTTGGAGAG FL024 BTBD12-SLX4 ex10R GTGGGGCAGGAAGTGAGGGAGAGT 428 i10 FL057 BTBD12-SLX4 ivs10-1F CCAGTTTCAGACGGACAGC FL058 BTBD12-SLX4 ivs10-1R CCATGCCCCAGGAAAAG 998 i10 FL059 BTBD12-SLX4 ivs10-2F GTGTGGGCAGGAACTCAG FL060 BTBD12-SLX4 ivs10-2R TCACCCAGCAGGACACG 939 11 FL025 BTBD12-SLX4 ex11F GTAAGCCATGATCACACCACT FL026 BTBD12-SLX4 ex11R TGCAGCACAACCCTCCAG 413 i11 FL061 BTBD12-SLX4 ivs11-1F CACTATCTCTACACTGCGGACACT FL062 BTBD12-SLX4 ivs11-1R AAAAGACATGGCATCAGACACAAT 759 i11 FL063 BTBD12-SLX4 ivs11-2F AGGCAGCAGCTCAGAGGGACAAGT FL064 BTBD12-SLX4 ivs11-2R ACGCCAAACCTGCAACACGAAACA 944 12 FL047 BTBD12-SLX4 ex12-1F TGGGTGTTTCTGGCAAGGAGTGTG FL048 BTBD12-SLX4 ex12-1R CCAGGGAGCCAGGCGATGAGAAAC 915 12 FL049 BTBD12-SLX4 ex12-2F AGATGATGCCGGGGATTACGAACA FL050 BTBD12-SLX4 ex12-2R CCCGCTCCTGAGGCTGCTGATT 913 12 FL051 BTBD12-SLX4 ex12-3F GGGCTCCCTGGCTGTTCTGT FL052 BTBD12-SLX4 ex12-3R GCCCGGCTCTCCTCCACTG 1111 i12 FL065 BTBD12-SLX4 ivs12-1F GCCGGGCTGGTTCTCAT FL066 BTBD12-SLX4 ivs12-1R GACTTTGGTTCTTCTGCCTTTTAT 1344 i12 FL067 BTBD12-SLX4 ivs12-2F TCGGGAGGGTGACTAAGAAT FL068 BTBD12-SLX4 ivs12-2R CTGGGAGGTCGAGGCTACAAT 1261 i12 FL083 BTBD12-SLX4 ivs12-3F CAAGCTGGTCTCGAACTCCTAAC FL084 BTBD12-SLX4 ivs12-3R GTGAAACCCCGTCTCTATT 1217 i12 FL071 BTBD12-SLX4 ivs12-4F GGCCACCACACCCTGCTAACTTTT FL072 BTBD12-SLX4 ivs12-4R TCTTCAGCACCGGCGTCTCCATAA 1068 13 FL031 BTBD12-SLX4 ex13F CCAGACAGGGAAGGGGTGAC FL032 BTBD12-SLX4 ex13R TGGGGAATGGTACGCAGAGA 486 i13 FL073 BTBD12-SLX4 ivs13-1F ACTGGATAGGTTGGCGGTCTTC FL074 BTBD12-SLX4 ivs13-1R CCAGCTACTAGGGAGGATGAGGT 893 i13 FL075 BTBD12-SLX4 ivs13-2F TGGGAGCAGCATATCTTTTGTT FL076 BTBD12-SLX4 ivs13-2R AGCGGCTGTGAGGACTGG 645 14_15 FL033 BTBD12-SLX4 ex14_15F TAAGATAGGGAACGTGGAGTGTGG FL034 BTBD12-SLX4 ex14_15R ATTGGGCTGTGGTCATCATCA 1399 i5 FL086 BTBD12-SLX4 i5-3R seq 2076_2096 TTGGGCTGTGGGGAAAGAATA i9 FL087 BTBD12-SLX4 i9-1R seq 509_527 ATGCAAATGGGAGGAGGTC i10 FL085 BTBD12-SLX4 i10-2R seq 1275_1295 CCTCCTCGGTTCCAGCGTATT 2 621 slx4 413F CCCCCGCCTCGTCCTCCAAAAG 2 641 slx4_1051R TGGAGCAGAGGCAAGCACACC 5 640 slx4_1698R GGGGGCCAACCTCCATCTTCACA 7 627 slx4_2055F TGAAACCACAGGCCGACAGATAGA 8 639 slx4 _2386R CAGTGGGGGTGCCGTGGAG 10 628 slx4 2647F CTCGGGCTGCTGGTTGCTGACTTT 12 629 slx4 3310F CAGGTGCAGAAACAGTGGGACAA 12 623 slx4 3910F CCAGGGCACCAGAAAGGCAAAGA 12 630 slx4 4516F GGAAACAGGGAAGGGAACGAAGTC 15 634 slx4 5791R CTCAAATGCCGCTCCAAACTCACA 15 633 slx4 6354R GGAAGGGGCTGGAGTCTGTAAATC 15 624 slx4 6804R GTGGGCTCAGAGGGTGGCAAATGT

Nature Genetics: doi:10.1038/ng.750 Supplementary Table 2. Mutagenesis primers SLX4_C296A_C299A GGAAAAGGGTTTGTTCTTCGCCCAGATTGCTCAAAAG SLX4_C336A_C339A CCTCAGATCCCTGAGGCCCCGATTGCTGGGAAACCGTT

Supplementary Table 3. siRNA sequences SLX4_1 TTTGGATGAAGATTTCTGAGATCTG, SLX4_2 TTCCGTGGCTCCTTCTTGCTGGTGG SLX4_3 AAGAGTTCCTGGAAATTCTCGGCCC FANCI (used as a combination of three at total concentration of 50 nM) TCTCCTCAGTTTGTGCAGATGTTAT, GGCAGCTGTGTGGACACCTTGTTAA GCTGGTGAAGCTGTCTGGTTCTCAT ATR (used as a combination of three at total concentration of 50 nM) GGGAAATAGTAGAACCTCATCTAAA GGTCTGGAGTAAAGAAGCCAATTTA CCACCTGAGGGTAAGAACATGTTAA

Nature Genetics: doi:10.1038/ng.750