HSSB1 and hSSB2 Form Similar Multiprotein Complexes That Participate in DNA Damage Response

Yutong Xue,Yongjiang Li,Rakesh Kumar,Weidong Wang,Derek J. Richard,Parameswary A. Muniandy,Emma Bolderson,Michael Seidman,Kum Kum Khanna,Tej K. Pandita

doi:10.1074/jbc.c109.039586

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.

Highlights

Oligonucleotide/oligosaccharide binding fold (OB-fold)2 is a single strand DNA or RNA binding domain that has been found in proteins from all species
Cells depleted of hSSB1 display increased genomic instability, hypersensitivity to radiation, deficiency in activation of ATMdependent checkpoint pathway following DNA damage, and reduced efficiency in homologous recombination (HR)dependent repair of DNA double strand breaks (DSBs)
HSSB2 Forms a Complex with INTS3 and hSSBIP1 (C9ORF80)—Based on sequence similarity between the OBfold domains of hSSB2 and RMI2, we predicted that hSSB2 may resemble RMI2 as part of a multiprotein complex that participates in DNA damage responses

Summary

Introduction

Oligonucleotide/oligosaccharide binding fold (OB-fold)2 is a single strand DNA or RNA binding domain that has been found in proteins from all species. These three polypeptides were found to be hSSB2, INTS3, and hSSBIP1 by both mass spectrometry (data not shown) and immunoblotting (Fig. 1C, lanes 13–15). Reciprocal IP with INTS3 and hSSBIP1 antibodies yielded these two proteins and hSSB2, as revealed by immunoblotting (Fig. 1C, lanes 4 – 6 and 7–9).

Results

Conclusion