1. Structural Basis of SOSS1 Complex Assembly and Recognition of ssDNA
Wendan Ren, Hongxia Chen, Qiangzu Sun, Xuhua Tang, Siew Choo Lim, Jun Huang, Haiwei Song 
Cell Reports 
Volume 6, Issue 6, Pages (March 2014)
DOI: /j.celrep
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2. Cell Reports 2014 6, 982-991DOI: (10.1016/j.celrep.2014.02.020)
Copyright © 2014 The Authors Terms and Conditions

3. Figure 1
Structures of the SOSS 1 Complex in Isolation and in Complex with ssDNA
(A) Schematic representation of the domain organization in SOSSA, SOSSB1, and SOSSC.
(B) Structure of the SOSSAN/B1/C complex.
(C) Structure of the SOSSAN/B1/C complex with the orientation relative to (B) rotated along y axis by 180° then along x axis by 270°.
(D) Structure of SOSSAN/B1 complexed with dT12.
(E) Structure of SOSSAN/B1/C complexed with dT35. For (B), (D), and (E), structures are superimposed at SOSSAN to make sure that the orientations are identical. The N-terminal, linker, and C-terminal regions of SOSSAN are colored in green, gray, and yellow-green, respectively. SOSSB1 and SOSSC are in cyan and orange, respectively. The bound ssDNA is shown in pink.
Cell Reports 2014 6, DOI: ( /j.celrep )
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4. Figure 2 The Interfaces of the SOSSAN/B1/C Complex
(A) The first interaction area includes N-terminal loop of SOSSA and the helix α1 strand β6 of SOSSB1.
(B) The second interface involves α17 and α18 of SOSSA and the α1-β5 loop strand β1 as well as C-terminal tail of SOSSB1.
(C) The interface between SOSSA and SOSSC includes loop α13-α14, helices α14, α23 of SOSSA, and strands β1 and β2 as well as the loop β1-β2 of SOSSC. The color scheme for individual subunits is as in Figure 1. The key residues of the interfaces are shown in stick and labeled.
(D) The L42A, D435A, and R439A mutants of SOSSA fail to interact with SOSSB1 and SOSSC, respectively. 293T cells were transiently transfected with plasmids encoding Myc-tagged SOSSB1 or SOSSC together with plasmids encoding SFB-tagged wild-type SOSSA or its point mutants. Cell lysates were immunoprecipitated with anti-Flag antibody, and western blot analysis was performed with anti-Flag and anti-Myc antibodies.
(E) The E97A, F98A, and E104A mutants of SOSSB1 fail to interact with SOSSA. 293T cells were transiently transfected with plasmids encoding SFB-tagged SOSSA together with plasmids encoding Myc-tagged wild-type SOSSC or its point mutants. Cell lysates were immunoprecipitated with anti-Flag antibody, and western blot analysis was performed with anti-Flag and anti-Myc antibodies.
(F) The L95A and P99A mutants of SOSSC fail to interact with SOSSA. Coimmunoprecipitation experiments were performed similar to those described in (D).
Cell Reports 2014 6, DOI: ( /j.celrep )
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5. Figure 3
SOSSA Regulates SOSSB1/SOSSC Stability and Nuclear Localization
(A–C) SOSSA regulates SOSSB1/SOSSC stability. Cells to express wild-type SOSSB1/SOSSC (SiR-WT) or their point mutants defective in SOSSA binding (L95A, P99A, E97A, F98A, and E104A) under the control of a tetracycline-inducible promoter were generated. After 24 hr of doxycycline (1 μg/ml) induction, the cells were collected, and whole-cell lysates were probed with the indicated antibodies.
(D–F) SOSSA controls the SOSSB1/SOSSC nuclear localization. Cells to express wild-type SOSSB1/SOSSC (WT) or their point mutants defective in SOSSA binding (L95A, P99A, E97A, F98A, and E104A) under the control of a tetracycline-inducible promoter were generated. Cells were induced by doxycycline addition for 24 hr before fixing and processed for immunofluorescence.
Cell Reports 2014 6, DOI: ( /j.celrep )
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6. Figure 4
The Assembly of the SOSS1 Complex Is Necessary for Proper DNA Repair
(A) Cells that express siRNA#1-resistant wild-type SOSSA (SiR-WT) or its point mutants defective in SOSSB1 or SOSSC binding (SiR-L42A, SiR-D435A, and SiR-R439A) under the control of a tetracycline-inducible promoter were generated. The resulting cell lines were transfected twice with SOSSA siRNA. Twenty-four hours after the second transfection, cells were induced by doxycycline addition for 24 hr prior to IR (10 Gy) treatment. Six hours later, cells were fixed and processed for RAD51 immunofluorescence.
(B) Cells that express siRNA#1-resistant wild-type SOSSB1 (SiR-WT) or its point mutants (SiR-W55A, SiR-F78A, SiR-E97A, SiR-F98A, and SiR-E104A) under the control of a tetracycline-inducible promoter were generated. The resulting cell lines were transfected twice with SOSSB1 siRNA. Twenty-four hours after the second transfection, cells were induced by doxycycline addition for 24 hr prior to IR (10 Gy) treatment. Six hours later, cells were fixed and processed for RAD51 immunofluorescence. More than one hundred cells were counted in each experiment.
(C) Cells that express siRNA#1-resistant wild-type SOSSC (SiR-WT) or its point mutants defective in SOSSA binding (SiR-L95A and SiR-P99A) under the control of a tetracycline-inducible promoter were generated. Immunofluorescence staining was performed similar to those described in (A).
(D–F) Cells that express siRNA-resistant wild-type SOSSA/SOSSB1/SOSSC or their point mutants under the control of a tetracycline-inducible promoter were generated. The resulting cell lines were transfected twice with control or SOSSA/SOSSB1/SOSSC siRNAs. Following IR treatment, cells were permitted to grow for 14 days before staining. Experiments were done in triplicates. Results shown are averages of three independent experiments.
Cell Reports 2014 6, DOI: ( /j.celrep )
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7. Figure 5 The Interaction of SOSSB1 with ssDNA
(A) The bound ssDNA and the residues involved in the SOSSB1-DNA interactions are shown in stick models. The color scheme of protein is as in Figure 1, and the DNA is in gray.
(B) Mutational effects of SOSSB1 on the binding to dT48 examined by the electrophoretic mobility shift assay (EMSA). The mutant SOSSAN/B1/C complexes were reconstituted by mixing the SOSSB1 mutants with corresponding wild-type SOSS subunits and incubating on ice for 1 hr 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.
Cell Reports 2014 6, DOI: ( /j.celrep )
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