HSSB1 and hSSB2 form similar multiprotein complexes that participate in DNA damage response

Abstract

hSSB1 (human single strand DNA-binding protein 1) has been shown to participate in homologous recombination (HR)-dependent repair of DNA double strand breaks (DSBs) and ataxia telangiectasia-mutated (ATM)-mediated checkpoint pathways. Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways. This was evidenced by findings that hSSB2-depleted cells resemble hSSB1-depleted cells in hypersensitivity to DNA-damaging reagents, reduced efficiency in HR-dependent repair of DSBs, and defective ATM-dependent phosphorylation. Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80). Cells depleted of INTS3 and hSSBIP1 also exhibited hypersensitivity to DNA damage reagents, chromosomal instability, and reduced ATM-dependent phosphorylation. hSSBIP1 was rapidly recruited to laser-induced DSBs, a feature also similar to that reported for hSSB1. Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes. Thus, our data demonstrate that hSSB1 and hSSB2 form two separate complexes with similar structures, and both are required for efficient HR-dependent repair of DSBs and ATM-dependent signaling pathways.

FIGURE 1.

hSSB1 and hSSB2 form two separate complexes with INTS3 and hSSBIP1 (C9ORF80). A, a silver-stained SDS gel showing major polypeptides immunoprecipitated by FLAG antibody from HEK-293 cells stably expressing FLAG-hSSB2. A control IP was done using cells that do not express FLAG-hSSB2 (Mock IP). B, a silver-stained gel showing major polypeptides immunoprecipitated by a polyclonal antibody against hSSB2 from HeLa nuclear extract. Arrows indicate the three components of the hSSB2 complex that have been identified by mass spectrometry. C, immunoblotting to show co-IP of hSSB complex components from HeLa nuclear extract. A control IP using rabbit preimmune serum was included (Mock IP). The nuclear extract (Load), flow-through (FT), and eluted fractions are indicated at the top. D, a silver-stained SDS gel showing major polypeptides immunoprecipitated by an INTS3 antibody from Superose 6 fractions of HeLa extract that are enriched of hSSB complexes (fractions 34–38, see panel E). E, immunoblotting shows that components of hSSB complexes co-fractionate near fraction 36 on a Superose 6 gel filtration column. BRG1, a component of 1-MDa SWI/SNF (switch/sucrose nonfermenting) complex, was included as an internal control. The fractions corresponding to calibration proteins with known molecular mass are indicated at the bottom. F, immunoblotting to show that the levels of various hSSB complex components in NFF cells were reduced by siRNA depletion of its partners, indicating that hSSB complex components are interdependent for their stability. Immunoblotting of actin was used as loading control. G, graphic presentation showing quantification of the immunoblotting data in F.

FIGURE 2.

hSSB complexes are important in responding to DNA damages and maintaining genome stability. A and B, cell survival assays show that HeLa cells depleted of hSSB components are hypersensitive to ionizing radiation (A) and camptothecin (B). Error bars indicate S.E. C, depletion of INTS3 or hSSBIP1 results in increased spontaneous and IR-induced chromosomal aberration. Chromosomal aberrations were analyzed at metaphase in 293 cells. Cells in the exponential phase were irradiated with 2 Gy. Metaphases were harvested at 4 h after irradiation, and chromosomal aberrations were scored. Cells with INTS3 or hSSB1P1 knockdown showed significant differences in chromosomal aberration frequencies when compared with control cells (p > 0.05, Student's t test). Ataxia telangiectasia (A-T) cells were included as positive control. D, a graph shows results from an HR-dependent DSB repair assay for DR-U2OS cells depleted of various hSSB complex components. DR-U2OS cells depleted of BRCA2 and ATM were included as positive control. E, indirect immunofluorescence shows that hSSBIP1 was recruited to a DSB site induced by laser where it colocalizes with γH2AX. The recruitment occurs within 15 min of the laser treatment. The nuclear DNA was indicated by 4′,6-diamidino-2-phenylindole (DAPI) staining.

FIGURE 3.

Both hSSB1 and hSSB2 complexes are required for efficient ATM-dependent phosphorylation events in response to ionizing radiation. A, immunoblotting shows that phosphorylation of ATM itself and several of its substrates (indicated by circled P) in NFF cells was reduced by depletion of hSSB complex components. Cells were either unexposed or exposed to 6 Gy of ionizing radiation, and cell extracts were immunoblotted with anti-phosphorylation site-specific antibodies for ATM (Ser-1981), NBS1 (Ser-343), Chk1 (Ser-317), and Chk2 (Thr-68). ATM and actin were included as controls for equal protein loading. B–D, graphic representation shows quantification of the results in panel A. The result shown is a representative from two independent experiments that yielded reproducible data.