Journal of Cell Science ol epopoyae yAMadrglt ATM-dependent regulate and hSSB1 ATM damage, by DNA al., of phosphorylated to depletion et be response Thus, Richard could In 2009b). 2009; repair. al., DSB et al., impairs Zhang et focus hSSB1 2009; Li al., 2009; regulates et Skaar al., BRCA1 and 2008; et as such (Huang hSSB1 sites proteins, RAD51 repair damage, and damage damage DNA DNA DNA other of polypeptide Following formation to 211-residue N-terminus. relocates a the quickly is at hSSB1 fold domain (OB) 2008). oligonucleotide/oligosaccharide-binding repair an HR al., containing during role et import an (Richard plays (hSSB1) 1 protein binding 2008; Williams Paull, 2003; West, and 2007; al., 2008). al., Hopkins et et al., Takeda Nicolette et 2008; 2010; 2008; al., al., et Symington, Niu and 2010; et Mimitou Gravel 2010; Huertas, 2010; (Cejka recombination al., and replication et the 5 invade for 3 to chromatid a RAD51 a by sister generating by motorized the site, is which broken initiated is (ssDNA), the DNA DNA is It of stranded sides DNA. pathway homologous both genomic HR at undamaged broken resection the It the an that repair 2004). of to believed Bartek, template presence and as Lukas the sequence 2010; on Elledge, relies and Ciccia 2004; o:10.1242/jcs.132514 doi: 4850–4855 126, ß Science Cell of Journal 2013 July 15 Accepted H)rpi sa ro-retp frpi htesrsgenomic ensures that Go repair and of type (Aguilera 2004). error-free stability an al., recombination is homologous et repair pathways, Sancar (HR) repair and 2002; DSB Jackson, (Harper these and Among pathways and Rouse most conserved fusion them 2007; repair Elledge, evolutionarily loss, the and sense or activating chromosomal can are cells by cause errors occur, (DSBs) DSBs can Once replication DNA translocation. they breaks of as by double-strand types deleterious induced different DNA those damage damage, Among various hazards. DNA by environmental challenged of constantly is genome types our of integrity The Introduction hSSB1–INTS recombination. the homologous Moreover, correlated sites. the damage and DNA affinity sites the damage protein DNA to a Using at relocates words: forms C9orf80. BRCA1 Key INTS6 INTS6 and complex. and hSSB1, RAD51 hSSB1 and INTS3 of core INTS3 accumulation the hSSB1, with the of contains along subunit regulates major complex complex response, a of damage hSSB1 both as maintenance DNA hSSB1 core (INTS6) the and the 6 and the INTS3 response subunit with that damage complex complex DNA integrator shown stable the identified in been have role we has important purification, an It plays stability. (hSSB1) 1 genomic protein DNA-binding single-stranded Human Summary Drive, Center Medical W. 1150 School, ( Medical USA correspondence Michigan 48109, for of MI University *Author Medicine, Arbor, Internal Ann of MSRBII, Department Genetics, 5560 Yu* and Medicine Xiaochun Molecular of and Division Ma Teng Zhang, Feng DNA the in response participates damage complex hSSB1–INTS core A 4850 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. rvoseiec hw hthmnsnl-taddDNA- single-stranded human that shows evidence Previous N aaersos,hS1IT ope,Hmlgu recombination Homologous complex, hSSB1–INTS response, damage DNA [email protected] ´ mez-Gonza ) ´ e,20;Bre tal., et Bartek 2008; lez, nvitro in 9 and o3 to 9 nvivo in single- 9 end nti ope,IT6drcl neat ihIT3 nrsos to response In INTS3. with interacts directly INTS6 complex, this In . NS eoae oteDAdmg ie n regulates Discussion and and and Results sites RAD51 stabilizing damage sites. damage direct by DNA DNA at (HR) through RPA the INTS6 recombination induction, response to damage homologous complex. DNA damage relocates to response hSSB1–INTS DNA INTS6 In INTS3. the the with another interaction as in in INTS6 complex Zhang identified participates the 2009; we subunit in al., study, INTS3 et this important of Skaar In function 2009; elusive. the al., remains repair However, et HR 2009b). in Li al., participate 2009; et and al., sites 2009; et C9orf80 damage al., and (Huang DNA INTS3 et both to Skaar hSSB1, recruited N- 2009; to are its al., Similar 2009b). et through al., Li C9orf80 et 2009; and Zhang interacts al., hSSB1 directly et of (Huang it a domain terminus is and fold INTS3 residues OB whereas the 1043 complex, with with the protein in larger adapter much an as and a polypeptide considered form 104-residue is to a al., C), is subunit C9orf80 et complex complex. SOSS multi-subunit Zhang as was (INTS3) known 3 2009; (also hSSB1 subunit C9orf80 complex al., purifications, and integrator al., with et affinity associate et to protein Skaar identified Li initial 2008; 2009; From al., al., 2009b). et et (Huang Richard activation 2009; checkpoint cycle cell rvossuishv hw htIT6i uui nthe in subunit a is INTS6 S1A). that Fig. shown material have supplementary studies 1A; (Fig. Previous major INTS3 another is of DDX26a) as partner known that revealed (also analysis INTS6 spectrometry hSSB1, Zhang Mass besides bait. 2009; in the purification as exist al., affinity INTS3 may et tandem using subunit(s) unbiased Skaar performed other we 2008; whether 2009; complex, examine al., this al., To et (ATM) 2009b). et Richard al., to (Huang 2009; et kinase DSBs al., shown following et mutated repair Li been HR Ataxia-telangiectasia and has activation in complex complex participate hSSB1–INTS hSSB1–INTS3–C9orf80 the in subunit The major a is INTS6 hr Report Short Journal of Cell Science nta uiiainrsls epromdrvrepurification our reverse validate performed To INTS6. we and results, (supplementary hSSB1 purification INTS6 our on 2009b), initial and al., focused et Zhang hSSB1 mainly is 2008; al., study of genes et respectively. (Richard DDX26b that S1B) INTS6, Fig. and material than hSSB2 that and lower the hSSB2 complex hSSB1 of much identified transcription of integrator the also paralogs the Because we DDX26b, of Meanwhile, and INTS3. subunits with al., did other we associate et purification, stringent identify (Baillat the Using hSSB1 not 2009). al., with et associates Skaar 2005; that complex integrator indicated the with analyzed and Gy lysed 10 anti- control. were or with 0 cells blot with IR, A treated after antibodies. were hour cells One INTS3 293T between IR. DSBs. interaction of of The independent (D) An is control. (input). IP INTS6 blotted the and also as used were was lysates IgG cell irrelevant by anti-hSSB1 Whole analyzed anti-INTS3, antibodies. then with anti-INTS6 and blotting or buffer western and NETN and INTS3 with (IP) with lysed immunoprecipitation interacts were INTS6 cells (C) table. 293T of the results hSSB1. in The shown spectrometry. SDS- mass are 12% and coverage by staining peptide separated silver subjected by were were visualized elutes (Ctr) and Final control PAGE purifications. or streptavidin- (B) affinity and INTS6 tandem or tag, for (A) FLAG INTS3 tag, tag) (S peptide SFB-tagged binding hSSB1. expressing and stably INTS3 cell with 293T associates INTS6 1. Fig. b atnatbd a sda rti loading protein a as used was antibody -actin AB ellstsof lysates Cell (A,B) SB–NScmlxi h N aaersos 4851 response damage DNA the in complex hSSB1–INTS eadeso h Rtetet ugsigta sebyo the 1D). of (Fig. damage assembly DNA that of suggesting independent treatment, is were constitutively IR complex occurs Lysates the INTS6 of (IR). and that regardless found INTS3 radiation We between co-IP. ionizing interaction reverse damage and the of co-IP to DNA subjected Gy were and Cells collected 10 whether INTS6. with and INTS3 investigated treated between interaction we the regulates response, damage and 1C). INTS3 (Fig. we with co-IP. associates endogenously indeed antibody, hSSB1 reverse INTS6 that this and showed results specifically Using The (co-IP) INTS6 S1D). co-immunoprecipitation endogenous Fig. performed suggesting recognizes material protein, INTS6 antibody (supplementary this of a of our knockdown expression with that the Moreover, against polypeptide suppressed kDa. antibody siRNA 100 a by of anti-INTS6 recognized mass an which molecular generated INTS6, first endogenous that We complex. indicating hSSB1–INTS3 hSSB1. the in S1C), subunit INTS6 major Fig. with a indeed associated material spectrometry is were INTS6 mass supplementary hSSB1 by 1B; and confirmed (Fig. and INTS3 bait, that the analysis, as INTS6 using neato ihIT6 ssoni i.2,teN-terminal the 2D, Fig. in shown As INTS6. with C- interaction a the form that to other suggest each with results complex. interact also INTS3 stable these and is material INTS6 together, of that (supplementary terminus Taken orthologous region S2D). DDX26 conserved Fig. other highly INTS6 in a INTS3 both deletion present contain with Interestingly, C-terminal interaction DDX26b S2C). 2C). the its (Fig. and Fig. INTS6, abolished material INTS3 of also (supplementary with DDX26b mutant mutant of D8 interaction deletion mutant the C-terminal the to the abolished Similar D8, INTS6, that material found INTS6 (supplementary of We of folding S2A). protein mutants protein–protein predicted Fig. deletion the internal a on with generated as based interacts also INTS6 serve of we region could INTS3, which S2B), examine region To also motif. Fig. this binding is material INTS3 that of (supplementary suggesting INTS3 region conserved C-terminal of to the Similar evolutionarily acids INTS6. region, and amino N-terminal INTS3 the 881–1042 between interaction C-terminal the 2B, deletion examine abolished the Fig. to in internal shown of structure As of folded INTS6. deletion with its Fig. interacts series directly to material region a according which (supplementary INTS3 generated of region we mutants are hinge Therefore, that small areas S2A). a well-folded al., we by two analysis et contains folding linked Skaar protein INTS3 2009; the that From al., 2009b). determined et al., et (Huang that Zhang hSSB1 2009; indicating with 2A), interacts (Fig. hSSB1. INTS3 and hSSB1 INTS6 between and interaction the INTS3 INTS3 mediates between or INTS3 interaction the INTS6 affect not depletion and did However, INTS6 INTS6. or hSSB1 and of hSSB1 association abolished between treatment the association siRNA the specific examine by INTS6 INTS3 and of use and downregulation INTS3 we hSSB1 down assembled, is between knock complex to hSSB1–INTS the siRNAs how study C-terminus To the to binds INTS6 directly of INTS3 of C-terminus The eas ohhS1adIT3priiaei h DNA the in participate INTS3 and hSSB1 both Because and INTS3 INTS6, between interactions the examined next We ete xmndwihrgo fhS1wsrqie o its for required was hSSB1 of region which examined then We of N-terminus the that demonstrated have others and We nvivo in ssoni i.2A, Fig. in shown As . Journal of Cell Science ula xrcso 9Tclsuigsize-exclusion using and INTS3 cells hSSB1, 3A, 293T Fig. in of shown As extracts chromatography. nuclear oivsiaewehredgnu SB,IT3adINTS6 complex and INTS3 stable hSSB1, a endogenous form whether investigate To hSSB1 INTS3, with complex and stable a forms INTS6 hSSB1 between is it association INTS3, the binds of INTS6. mediates N- directly and INTS3 mutant the hSSB1 that Because of deletion domain probable INTS6. C-terminal fold with OB interaction the terminal the not disrupted but hSSB1, mutant, deletion 4852 nvivo in ora fCl cec 2 (21) 126 Science Cell of Journal nvivo in eaaye hs rtisin proteins these analyzed we , nvitro in htol h -emns u o h -emnso INTS3, of N-terminus the not found but we with C-terminus, cells, with the Sf9 co-expressed in associate only was C-terminus to that SFB–INTS6 or found N-terminus When were GST–INTS3 3B). INTS6 (Fig. and INTS3 glutathione hSSB1 using both down glutatione (streptavidin-binding pulled beads, and was INTS3 SFB-INTS6 When SBP-tagged (GST)–INTS3. hSSB1, tag) encoding peptide baculoviruses enx netgtdteascainbtenhS1 INTS3 hSSB1, between association INTS6 the and investigated a next to we corresponding fractions lysate of cell mass molecular in co-existed INTS6 odsettepoenpoenitrcinwti h complex, the within interaction protein–protein the dissect To nvitro in ltigwt h niae antibodies. western indicated and the IP with by blotting between examined interactions were The INTS6 cells. hSSB1and 293T hSSB1 in of expressed mutants were INTS3. deletion with interacts and hSSB1 wild-type of antibodies. Myc-tagged domain indicated fold the OB with The blotting (D) western examined and were IP INTS3 by The and cells. INTS6 293T between in interactions expressed their were and mutants DDX26b deletion INTS3. or with INTS6 interacts wild-type INTS6 FLAG-tagged of antibody. C-terminus anti-HA The with (C) blotting were western proteins by INTS3 examined exogenous The of antibodies. levels indicated expression Cell the cells. using 293T analyzed in were expressed lysates were right) the on shown mutants (as deletion with and interacts INTS3 wild-type INTS3 HA-tagged indicated of INTS6. the C-terminus with The blotting (B) western antibodies. were and lysates IP Cell by siRNA. analyzed hSSB1 control, or with treated INTS6 were INTS3, cells U2OS hSSB1. and and INTS6 INTS6 of regions interaction INTS3. the Mapping 2. Fig. , f netclswr oifce with co-infected were cells insect Sf9 . 0 to 300 ( A NS eitsteitrcinbetween interaction the mediates INTS3 ) , 0 kDa. 500 S -transferase Journal of Cell Science E ceai fdrc idn fteIT3wt NS n SB.N, hSSB1. and INTS6 with INTS3 terminus. the C antibodies. of C, indicated binding N-terminus; the direct with of virus blotting Schematic of western (E) combinations by or examined individual with and cells infection recombinant Sf9 Each from (B–D) harvested blotting hSSB1. was western and protein indicated INTS6 by INTS3, the analyzed against from and antibodies eluted SDS-PAGE using Proteins by column. separated filtration were gel fractions 6 Superose a on vivo oevr elto fIT3b iN significantly siRNA S4A). by Fig. INTS3 material INTS6. 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INTS3 (Fig. the of INTS6 C-terminus to that the and hSSB1 confirm to S3). with Fig. interacts further assay material (supplementary results pull-down INTS3 INTS6 of These GST of C-terminus C-terminus the the the between and performed INTS3 interaction direct also of the binds confirm We N-terminus directly INTS3 INTS6. direct the of C-terminus to that the little and 3D). confirm hSSB1 (Fig. Moreover, with INTS6 interacts further and 3C). results (Fig. hSSB1 between These INTS6 detected was to interaction bound directly hSSB1 INTS3, with complex stable a forms INTS6 3. Fig. . A 9Tcl xrcswr nlzdb ieecuinchromatography size-exclusion by analyzed were extracts cell 293T (A) c 2X(un ta. 09 ie al., et Li 2009; al., et (Huang H2AX nvitro in SB–NScmlxi h N aaersos 4853 response damage DNA the in complex hSSB1–INTS and in N ein hog t neato ihIT3adhSSB1. and to INTS3 recruited with is interaction S4C). INTS6 its that Fig. through lesions suggest material DNA results (supplementary 4B), these (Fig. hSSB1 together, IRIF or IRIF the Taken affect INTS3 INTS6 not did either of DDX26b of and formation INTS6 supplementary interaction of impaired depletion Depletion the whereas and IRIF. also INTS6 that hSSB1 of 4B suggest formation (Fig. of for results important is INTS6 These INTS3 with S4B). of Fig. IRIF material the decreased F-oiieclswseaie yfo yoer.Dt r averages are Data cytometry. of flow percentage by ( The examined adeno-I-SecI. was The by cells siRNAs. infected GFP-positive indicated then the were HR with cells impairs treated siRNA-treated the DDX26b were with and cells co-stained INTS6 U2OS-DrGFP and irradiation of with repair. after irradiated Knockdown hours then (D) 6 and antibodies. fixed hours indicated were with Cells 48 treated IR. for of were siRNA Gy cells DDX26b 10 U2OS and RPA. 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NP-40, were of stably Cells (0.5% partners PBS. cells buffer with binding NETN 293T washed and for described of harvested was search were cultures INTS6 tags) To liter SBP 2009a). 1 and al., FLAG INTS6, (S, et protein (Zhang tagged previously triple Signaling, SFB of chromatography Cell Purification size-exclusion and 2009b). and purification al., Protein et (ab95083) (Zhang RAD51 described and previously Abcam as anti-phospho-H2AX obtained anti-hSSB1, from were antibodies anti-INTS3, purchased Rabbit respectively. were antibodies b ENbfe otiig1 MNFad5 Mglycerophosphate. mM 50 and standard 2009a). NaF following al., et performed (Zhang mM were previously 10 described blotting as protocol western containing and Immunoprecipitation buffer NETN ed Nvgn.Tebudpoen eete ltdwt D apebuffer, sample spectrometry. 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J. Harper, E., M. Sowa, K., Hurov, III, R., E. McDonald, C., Cotta-Ramusino, ica .adElde .J. S. Elledge, and A. Ciccia, tiig el iha es he la oiwr osdrda oipstv cells. foci-positive as considered were foci clear we three non-specific foci, avoid least To damage-induced at cells. 500 with DNA count cells and quantify slide staining, each To from fields specified. nine choose for usually recovery otherwise IRIF, by followed examine unless Gy, To 10 hours, with glass microscope. 4 irradiated fluorescence onto and coverslips a mounted on cultured using were were visualized coverslips cells incubated cells The and the minutes. times and with 30 three slides incubated for washed then antibody were and secondary Samples minutes. serum with 60 goat for 5% antibody with primary blocked were Samples temperature. ek,P,Cnao . oazk . auaSs,T,Pkae,S,Cmbl,J L. J. Campbell, S., Pokharel, T., Masuda-Sasa, P., Polaczek, E., Cannavo, P., Cejka, J. Lukas, and C. Lukas, J., Bartek, Na A., M. Hakimi, D., Baillat, Go and A. Aguilera, References Fund. at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.132514/-/DC1 Research online available Cancer material Supplementary months. Program 12 Ovarian after Schreiber Childhood release the Ann for of the PMC Research from of in for Deposited recipient Award Defense. Association a of is the Excellence Department T.M. of the Award. recipient from of Cancer a Award recipient Scholar a is is Hope F.Z. [grant X.Y. of Health X.Y.]. Era of to Institutes the CA130899 National and the CA132755 by numbers supported was work This manuscript. Funding T.M. the on and manuscript. commented the F.Z. and prepared project. discussed X.Y. the authors and All F.Z. designed experiments. and the conceived performed X.Y. and F.Z. contributions Author in summarized are reactions QPCR master PCR for Green S1. 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