Integrator3, a Partner of Single-stranded DNA-binding Protein 1, Participates in the DNA Damage Response

Single-stranded DNA-binding protein 1 (SSB1) plays an important role in the DNA damage response and maintenance of genomic stability. Here, by using protein affinity purification, we have identified Integrator3 (INT3) as a novel partner of SSB1. INT3 forms a complex with SSB1 by constitutively interacting with SSB1 regardless of DNA damage. However, following DNA damage, along with SSB1, INT3 relocates to the DNA damage sites and regulates the accumulation of TopBP1 and BRCA1 there. Moreover, INT3 controls DNA damage-induced Chk1 activation and G₂/M checkpoint activation. In addition, INT3 is involved in homologous recombination repair by regulating Rad51 foci formation following DNA damage. Taken together, these results demonstrate that INT3 plays a key role in the DNA damage response.The DNA damage response, including DNA damage checkpoint activation and DNA damage repair, ensures genomic stability under genotoxic stress. Among various types of DNA damage, DNA double-strand breaks (DSBs)³ are the most deleterious, easily causing chromosomal loss, fusion, and translocation. However, cells can sense and repair DNA DSBs by activating evolutionarily conserved pathways (1–3). Following DNA DSBs, ATM, ATR, and DNAPK, a family homologous to phosphoinositide 3-kinases (4, 5), are activated and phosphorylate histone H2AX at the DNA damage sites (6). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and TopBP1 (2, 7–9), at the DNA damage sites. Translocalization of these proteins to the DNA DSBs facilitates DNA damage checkpoint activation by activating downstream Chk1/Chk2 kinases, which arrest the cell cycle at G₁, S, or G₂ phase (10). In addition, it also enhances the efficiency of DNA damage repair by recruiting and stabilizing the DNA repair machinery at the DNA damage sites (11).Among these important mediators, single-stranded DNA (ssDNA)-binding proteins play important roles during the DNA damage response. For example, following DNA damage, the MRN complex recognizes DNA DSB ends and processes the blunt ends into ssDNA overhangs (12). The replication protein A (RPA) complex, a group of ssDNA-binding proteins, immediately coats these ssDNA overhangs and loads and activates the ATR·ATRIP complex at the DNA damage sites (13). Meanwhile, the RPA complex protects ssDNA from nucleolytic resection and facilitates Rad51 filament formation along ssDNA overhangs, which is a key step for homologous recombination repair (14). Moreover, RPA70 and RPA32 subunits in the complex could recruit several DNA damage response factors to the DNA damage sites that enhance the efficacy of DNA damage repair (15).Besides the RPA complex, several other ssDNA-binding proteins have been identified to participate in the DNA damage response recently. One of them is ssDNA-binding protein 1 (SSB1) (16). Human SSB1 is a 211-amino acid polypeptide with an N-terminal oligosaccharide/oligonucleotide-binding (OB) domain. It has been shown that SSB1 is phosphorylated by ATM and relocates to the DNA damage site following DNA DSBs. Loss of SSB1 impairs DNA damage-induced checkpoint activation and induces genomic instability. Like the RPA complex, SSB1 participates in homologous recombination by facilitating Rad51·ssDNA filament formation and stabilizing Rad51 at the DNA damage sites. Interestingly, SSB1 has a homolog SSB2 that contains an almost identical OB domain at the N terminus. However, the function of SSB2 in the DNA damage response is not clear yet.To examine the molecular mechanism and functional pathway of SSB1 and SSB2 in the DNA damage response, we have searched for functional partners of SSB1 and SSB2 by using protein affinity purification. We have found Integrator3 (INT3) to be a common partner of both SSB1 and SSB2. Like SSB1, following DNA damage, INT3 relocates to the DNA damage sites and regulates ATR activation. Moreover, INT3 not only participates in DNA damage checkpoint activation but also regulates homologous recombination repair. Taken together, we have found a novel mediator in the DNA damage response.