|Home | About | Journals | Submit | Contact Us | Français|
In this issue of Molecular Cell, Huang et al. (2009) describe two heterotrimeric single-stranded DNA binding complexes, SOSS1 and SOSS2, that function downstream of the MRN complex to promote DNA repair and the G2/M checkpoint.
Single-stranded DNA (ssDNA) binding proteins (SSBs) are essential for most DNA metabolism, including DNA replication, repair, recombination, and damage signaling. In addition to protecting ssDNA, these proteins participate in complex protein interactions that regulate polymerase and helicase activities, strand exchange reactions during homologous recombination (HR), and checkpoint kinase activation in the DNA damage response. The most thoroughly studied SSB in eukaryotes is the replication protein A (RPA) complex. RPA consists of three subunits, RPA70, RPA32, and RPA14 that contain multiple DNA binding and protein interaction domains joined by flexible linkers.
Recently, two new SSBs, hSSB1 and hSSB2, were identified in the human genome. hSSB1 and hSSB2 are conserved in metazoans and contain a single oligonucleotide/oligosaccharide-binding (OB) domain. hSSB1, like RPA, localizes to sites of DNA damage (foci), and silencing hSSB1 causes checkpoint defects, reduced HR, and increased radiosensitivity (Richard et al., 2008). Unlike RPA, hSSB1 does not localize to replication foci, and may not function in DNA replication.
In this issue of Molecular Cell, Huang et al. discover that hSSB1 and hSSB2 function at DNA double-strand breaks (DSBs) as part of heterotrimeric complexes with INTS3 and C9orf80 (Huang et al., 2009). They name the INTS3/hSSB1/C9orf80 and INTS3/hSSB2/C9orf80 complexes Sensor of Single-stranded DNA complex 1 and 2 (SOSS1/2). INTS3, hSSB1/2, and C9orf80 are designated SOSS subunits A, B1/2, and C, respectively (Figure 1). The assembly of the complexes is dependent on SOSS-A. Like RPA and hSSB1, SOSS-A silencing results in checkpoint and HR defects as well as hypersensitivity to ionizing radiation. Similar results are also reported by Li and colleagues (Li et al., 2009).
RPA-recruitment to ssDNA at a DSB is dependent on the resection of the DNA end catalyzed by Mre11/Rad50/Nbs1 (MRN) in cooperation with Sae2/CtIP (Limbo et al., 2007; Sartori et al., 2007). Strikingly, silencing MRN prevents both RPA and SOSS focus formation in S/G2 phase cells treated with ionizing radiation, but CtIP silencing only impairs RPA and not SOSS focus formation. SOSS can also form foci in G1 phase cells independently of the MRN complex. Furthermore, both CtIP and RPA localization are independent of SOSS. Thus, RPA and SOSS localization to DSBs are regulated by distinct mechanisms. Since CtIP and MRN are thought to work together to resect DSBs in S/G2 phase cells, it remains unclear how SOSS is recruited in G1 and if resection is required for recruitment in S/G2. Nonetheless, the differences between the MRN-CtIP-RPA and the MRN-SOSS pathways suggest that RPA and SOSS may have different functions at DSBs. Consistent with this conclusion, simultaneous silencing of SOSS-A and CTIP causes a greater HR defect than silencing either alone.
Of course, a number of questions remain. What is the functional relationship between RPA, SOSS1 and SOSS2, and why three distinct SSBs? What makes the SOSS complexes specific to DNA repair activities and not DNA replication? How does SOSS promote repair?
Although the SOSS1 and SOSS2 complexes share two subunits and the defining subunits (SSB1/2) share sequence similarity, they do not act redundantly since silencing of either yields checkpoint and repair defects (Li et al., 2009). Outside of the N-terminal OB-fold domain and a small 13 amino acid stretch at the C-terminus, SSB1 and SSB2 lack significant sequence similarity. This divergent region may impart unique activities to the SOSS1/2 complexes.
Thus far, the characterized functions of SOSS1/2 are limited to repair and signaling that happens at DSBs, in contrast to the essential activities of RPA in other processes including DNA replication. It is possible that differences in ssDNA binding limit SOSS to only a subset of the DNA metabolic processes with ssDNA intermediates. SOSS contains only a single OB-fold that presumably binds ssDNA. In contrast, RPA contains six OB-fold domains, four of which bind DNA. Therefore, SOSS is predicted to have lower affinity for ssDNA than RPA. Indeed, the measured affinity of SSB1 to ssDNA is approximately 100-fold less than the affinity of RPA, and there is no evidence that either SOSS-A or SOSS-C bind DNA. RPA also can bind DNA in at least three different conformations depending on the ssDNA length. It is likely that SOSS will have a much more restricted set of binding conformations. These factors may limit its access to ssDNA intermediates and may even create a requirement for another cofactor to promote SOSS-ssDNA at the expense of RPA-ssDNA. As yet, there is no direct evidence that ssDNA binding is even necessary for the function of the SOSS complex at DSBs. A more detailed analysis of the DNA binding activities of SOSS will help resolve these questions.
RPA activity in replication, recombination, and repair requires protein-protein interactions through four binding surfaces on the RPA70 and 32 subunits (Fanning et al., 2006). Like RPA, SOSS likely acts by interacting with other proteins in addition to DNA. Huang et al., describe an interaction between SOSS-A and Nbs1, but the significance of this interaction is unknown (Huang et al., 2009). It may regulate MRN or SOSS localization or perhaps checkpoint signaling. Silencing of SOSS subunits is reported to reduce the activity of the ataxia-telangiectasia mutated (ATM) checkpoint kinase (Li et al., 2009; Richard et al., 2008), and ATM phosphorylates hSSB1. If parallels with RPA are drawn, SOSS1/2 may contribute to ATM activation via MRN binding much like RPA regulates ATR via an interaction with ATRIP (Cimprich and Cortez, 2008). However, contributions of SOSS activity to ATR-ATRIP regulation have not been excluded.
In summary, Huang et. al., discovered two new metazoan SSB complexes, SOSS1 and SOSS2 that regulate DNA repair and checkpoint responses to DSBs. Further studies will be needed to define the precise functions of these SSB complexes in DNA repair and signaling and relate them to the other major SSB in eukaryotic cells, RPA.