Cell Reports, Volume 6

Supplemental Information

Structural Basis of SOSS1 Complex

Assembly and Recognition of ssDNA

Wendan Ren, Hongxia Chen, Qiangzu Sun, Xuhua Tang, Siew Choo Lim, Jun Huang, and

Haiwei Song

SUPPLEMENTARY EXPERIMENTAL PROCEDURES

Protein expression and purification

The N-terminal region of SOSSA (residues 1-500; SOSSAN) was cloned into the

pGEX6p-1 vector and the full length SOSSB1 was inserted into the MCS2 of

pACYCDuet-1. The resultant plasmids were co-transformed into E.coli BL21 (DE3)

strain. Proteins were expressed for 12 hr at 18 after induction with 0.1mM IPTG. Cells

were harvested, resuspended in lysis buffer (20mM MES pH6.1, 150mM NaCl, 2mM

DTT), and lysed by sonication. After centrifugation, the clarified cell lysate was

incubated with glutathione-Sepharose 4B beads and GST-SOSSAN/B1 was eluted with

elution buffer (20mM MES pH6.1, 150mM NaCl, 2mM DTT, 20mM reduced

glutathione). The GST tag was removed by PreScissionTM protease (GE healthcare), and

the released SOSSAN/B1 complex was further purified by glutathione-Sepharose 4B,

MonoS and Superdex 200 gel filtration columns uccessively (GE healthcare). The full

length SOSSC was cloned into the pGEX6p-1 vector and expressed as a GST fusion

protein in E.coli BL21 (DE3). SOSSC was purified using glutathione-Sepharose 4B and

MonoS columns using the same approach as described for SOSSAN/B1. Purified SOSSC

and SOSSAN/B1 were mixed at a molar ratio of 1.5:1, incubated on ice for at least 30

min, then the SOSSAN/B1/C complex was separated from SOSSAN/B1 and SOSS-C by

size-exclusion chromatography on a Superdex 200 column. The purified SOSSAN/B1 and

SOSSAN/B1/C complexes were concentrated to 25mg/ml for crystallization. The

Selenomethionine (SeMet)-substituted SOSSAN/B1 was expressed in a minimal medium

containing 20mg/L SeMet and purified in the same way as that used for the native protein

except 10mM DTT was used in the lysis buffer. All the mutants of SOSSA and SOSSB1

were cloned by splicing through overlap extension PCR (SOE-PCR). The mutated

SOSS1 complexes were purified using the same procedure as that used for the wild type

complexes.

Crystallization

All the crystals were grown by the hanging-drop vapor diffusion method at 16° .

Crystals of native and SeMet-substituted SOSSAN/B1/C were obtained by mixing equal

volumes of protein solution and a reservoir solution containing 21% PEG2000 MME,

0.1M MES pH6.0, 0.05M sodium chloride. To crystallize SOSSAN/B1/dT12 and

SOSSAN/B1/C/dT35, SOSSAN/B1 and SOSSAN/B1/C were mixed with 12-nucleotide

poly(dT) and 35-nucleotide poly(dT) respectively at a molar ratio of 1:1. Crystals of

SOSSAN/B1/dT12 were grown from a condition containing 16% PEG3350, 0.1M MES

pH6.0, 0.1M ammonium formate while crystals of SOSSAN/B1/C/dT35 were grown from

a reservoir solution consisting of 20% PEG2000 MME, 0.1M MES pH6.0, 0.05M sodium

chloride. All the crystals were cryoprotected by inclusion of 20% ethylene glycol or

glycerol in the reservoir solution and flash-frozen in liquid nitrogen.

Electrophoretic mobility shift assay

Native acrylamide electrophoretic mobility shift assay was used to detect the binding

affinity of the SOSS1 complex with a 48-nucleotide poly(dT) (dT48). Wild type

SOSSAN/B1 or their mutants were mixed with SOSSC at a molar ratio of 1:1 and

incubated on ice for at least 60min. Then each of the reconstituted protein complex at

concentrations of 1.8 fmol, 3.6 fmol and 7.2 fmol was incubated with 5 fmol 3’-biotin

labeled dT48 in a buffer (20mM Tris-HCl, pH7.5, 50mM NaCl, 1mM MgCl2, 0.5mM

EDTA, 0.2mM DTT) on ice for 60 min. The reactions were resolved on 5% native gel

with 0.5 TBE buffer and were electrophoretically transferred to Nylon membrane and

crosslinked at 120mJ/cm2 using a UV-light crosslinking instrument equipped with

254nm bulbs. The subsequent blocking, washing and detection were performed according

to the manufacturer’s instructions (Lightshift chemiluminescent EMSA Kit, Pierce).

Isothermal titration calorimetry

ITC measurements were performed at MicroCa VP-ITC (MicroCal Inc.). Protein

samples were dialyzed into a buffer containing 20mM HEPES (pH7.2), 150mM KCl and

1mM MgCl2. For analyzing the binding of dT12 to SOSSB1, SOSSA /B1, and

SOSSA /B1/C, 500μM dT12 were injected into the calorimetric cell containing 50μM

protein. For analyzing the binding of dT48 to SOSSB1, SOSSA /B1, SOSSA /B1/C and

SOSS4m/B1/C, 100μM dT48 were injected into the cell containing 12μM protein.

Titrations were all performed at 25° , initiated with 5μl injection, followed by twenty-

four 10μl injections with 300 seconds equilibration time between injections. The raw data

represented the heat signal (μcal/sec) versus time (minutes) and were analyzed using the

Origin 7.0 program, fitted to a single-site binding model.

Antibodies

Antibody specifically recognizing RAD51 was previously described (Huang et al.,

2009; Wan et al., 2013). Anti-Myc (9E10) and anti-Flag (M2) antibodies were purchased

from Covance and Sigma, respectively. Anti-GAPDH antibody was purchased from

Millipore.

Cell culture and transfection

293T and HeLa Cells were maintained in DMEM supplemented with 10% fetal

bovine serum and 1% penicillin and streptomycin. Cell transfection was performed using

Lipofectamine 2000 (Invitrogen), following the manufacturer’s protocol.

Constructs

The mammalian expression plasmids for SFB- or Myc-tagged SOSSA, SOSSB1 and

SOSSC were previously described (Huang et al., 2009). Site-directed mutagenesis was

performed according to standard procedures to obtain the SOSSA, SOSSB1, or SOSSC

mutants. For transient expression of SOSSA, SOSSB1, SOSSC or their mutants, the

corresponding fragment in the entry vector was transferred into Gateway compatible

destination vector which harbor either an N-terminal triple-epitope tag (S protein tag,

Flag epitope tag and Streptavidin binding peptide tag) or an N-terminal Myc tag. All

clones were sequenced to verify desired mutations.

RNA interference

All siRNAs were synthesized by Dharmacon Inc. The siRNAs were 21 base pairs

and sequences are as follows: SOSSA siRNA: 5’-GAUGAGAGUUGCUAUGACA

dTdT; SOSSB1 siRNA: 5’-CGACGGAGACCUUUGUGAAdTdT; SOSSC siRNA: 5’-

GAACCAGUCUUCAACAAAUdTdT and control siRNA: 5’-UUCAAUAAAUUC

UUGAGGUUU. The siRNA-resistant wild-type and mutant SOSSA constructs were

generated by changing 9 nucleotides in the SOSSA siRNA targeting region (G1569A,

T1572C, G1575A, A1576T, G1577C, T1578A, C1581T, T1584C, and C1587T

substitutions). The siRNA-resistant wild-type and mutant SOSSB1 constructs were

generated by changing 7 nucleotides in the SOSSB1 siRNA targeting region (G6A, G9A,

G12A, C15G, T18C, G21C and G24A substitutions). The siRNA-resistant wild-type and

mutant SOSSC constructs were generated by changing 6 nucleotides in the SOSSC

siRNA targeting region (C99T, G102A, T105A, A108T, A111T, and T114C

substitutions).The siRNA transfection was performed with 100 nM siRNA duplexes

using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer's instruction.

Transfection was repeated twice with an interval of 24 hr to achieve maximal RNAi

effect.

Co-immunoprecipitation and western blotting

For Flag immunoprecipitations, a 0.8-ml aliquot of lysate was incubated with 1 μg of

the Flag monoclonal antibody and 25 μl of a 1:1 slurry of Protein A Sepharose for 2 hr at

4°C. The Sepharose beads were washed three times with NTEN buffer, boiled in 2 X

SDS loading buffer, and resolved on SDS-PAGE. Membranes were blocked in 5% milk

in TBST buffer and then probed with antibodies as indicated.

Cell survival assays

HeLa cells were transfected twice with control siRNA or siRNAs specifically

targeting SOSSA, SOSSB1 or SOSSC. 24 hr after the second transfection, cells (1 X 103)

were splitted and transferred into 60 mm dishes. Cells were incubated for 24 hr before

they were treated with IR as indicated. The medium was replaced 24 hr later and cells

were then incubated for 14 days. Resulting colonies were fixed and stained with

Coomassie blue.

Lentivirus Packaging and Infection

Tet-On inducible SFB-tagged lentiviral vector and packaging plasmids (pMD2G and

pSPAX2) were kindly provided by Professor Songyang Zhou (Baylor College of

Medicine). SOSS entry constructs were transferred into the Gateway-compatible SFB-

tagged lentiviral vector. Virus supernatant was collected 48 hr after the co-transfection of

lentiviral vectors and packaging plasmids (pMD2G and pSPAX2) into 293T cells. Cells

were infected with viral supernatants with the addition of 8 μg/ml polybrene (Sigma), and

stable pools were selected with medium containing 500 μg/ml G418 (Calbiochem). The

expression of the indicated genes in the stable pools was induced by the addition of 1

μg/ml doxycycline (Sigma) for 24 hr for the experiments presented in this report.

SUPPLEMENTARY REFERENCES

Huang, J., Gong, Z., Ghosal, G., and Chen, J. (2009). SOSS complexes participate in the

maintenance of genomic stability. Mol Cell 35, 384-393.

Wan, L., Han, J., Liu, T., Dong, S., Xie, F., Chen, H., and Huang, J. (2013). Scaffolding

protein SPIDR/KIAA0146 connects the Bloom syndrome helicase with homologous

recombination repair. Proc Natl Acad Sci U S A 110, 10646-10651.

Table S1. Data collection and refinement statistics, related to Experimental

Procedures

Data collection SeMet-SOSSAN/B1/C SOSSAN/B1/C/dT35 SOSSAN/B1/dT12 Wavelength (Å) 0.9798 0.9798 0.9798 Resolution limit (Å) 2.0 2.3 2.3 Space group P3121 P212121 P21212 Cell parameters a/b/c(Å) 116.81/116.81/109.83 115.83/76.65/87.29 98.63/162.52/65.74 / / (°) 90/90/120 90/90/90 90/90/90 Unique reflections (N) 58840 37719 47747 Total Reflections (N) 698927 218092 371636 Mean (I/ ) 9.9 (2.4) 7.8 (2.7) 11.4(2.5) Completeness (%) 100.0 (100.0) 99.8 (99.8) 99.8 (99.8) Rmerge

a 0.174 (0.791) 0.145 (0.629) 0.110 (0.790)

Number of Se sites 15

Refinement Statistics

Data range (Å) 20.0-2.0 20.0-2.3 20.0-2.3

Used Reflections (N) 58732 37578 47624 Protein residues 577 615 580 Protein atoms 4608 4971 4735 Solvent molecules 410 213 159 RNA bases -- 6 9 Rwork

b(%) 17.19 17.99 18.80 Rfree

c (%) 19.53 21.83 22.69 R.m.s deviation Bond length (Å) 0.006 0.005 0.005 Bond angles (°) 0.990 0.928 1.017 Ramchandran plot (% residues)

Allowed 99.6 99.4 99.8 Generously allowed 0.4 0.2 0.0 Disallowed 0.2 0.4 0.2

Values in parentheses indicate the specific values in the highest resolution shell. aRmerge = |Ij-<I>|/ Ij, where Ij is the intensity of an individual reflection, and <I> is the average intensity of that reflection. bRwork = ||Fo| - |Fc||/ |Fc|, where Fo denotes the observed structure factor amplitude, and Fc denotes the structure factor amplitude calculated from the model.cRfree is as for Rwork but calculated with 5.0% of randomly chosen reflections omitted from the refinement.

Figure S1, related to Figure 1. Superposition of SOSSAN with the middle domain of eIF4G and

Paip1. The coloring scheme of N-SOSSAN and C-SOSSAN is as in Figure 1. The middle domain of eIF4G (eIF4Gm) and Paip1 are shown in purple and blue respectively

Figure S2, related to Figure 2. Structure-based sequence alignment of (A) SOSSA, (B) SOSSB1

and (C) SOSSC between different species. The secondary structural elements for these proteins are shown and numbered. The invariant residues and conserved residues are highlighted in red-shaded boxes and yellow-shaded boxes, respectively.

Figure S3, related to Figure 4. The assembly of the SOSS1 complex is necessary for RAD51 focus

formation. Quantification results were the average of three independent experiments and were presented as mean±SEM.

Figure S4, related to Figure 5. The interaction of

SOSSAN with ssDNA. (A) Electrostatic potential of SOSSAN , a symmetry-related SOSSAN molecule in the SOSSAN/B1/dT12 complex. The protein surface is colored according to the electrostatic potential generated by ABPS (blue as positive, red as negative, white as neutral). (B) The bound ssDNA and the residues involved in the SOSSA-DNA interactions are shown in stick models. The color scheme of protein is as in Figure 1 and the DNA is in grey. (C) Mutational effects of SOSSA on the binding to dT48 examined by EMSA. The mutant SOSSAN/B1/C complexes were reconstituted by mixing each of the SOSSAN mutants (R298A, H358A, S408A and M410A) with the corresponding wild type SOSS subunits and incubating on ice for 1 hour before loading to the gel. The amount of DNA shifted were quantified and normalized against the total DNA shifted by wild type SOSSAN/B1/C complex at the highest concentration. (D) The effect of quadruple mutations of Arg298A, His358A, Arg295A and Lys466A to Ala (SOSSA4m) on the binding of SOSSAN/B1/C to dT48 examined by ITC. SOSSA4m/B1/C was reconstituted by mixing SOSSA4m with SOSSB1and SOSSC. The upper panel shows the binding isotherm and the lower panel show the integrated heat for each injection fitted to a single-site model.

Figure S5, related to Figure 5. The binding of SOSSAN/B1, SOSSAN/B1/C and SOSSB1 to dT12

and dT48 examined by ITC. (A) Titrations of dT12 to SOSSAN/B1, SOSSAN/B1/C and SOSSB1. (B) Titrations of dT48 to SOSSAN/B1, SOSSAN/B1/C and SOSSB1. The upper panels show the binding isotherms and the lower panels show the integrated heat for each injection fitted to a single-site model.