Fanconi anemia (FA) is caused by mutations in 13 Fanc genes and renders cells hypersensitive to DNA interstrand cross-linking (ICL) agents. A central event in the FA pathway is mono-ubiquitylation of the FANCI-FANCD2 (ID) protein complex. Here, we characterize a previously unrecognized nuclease, Fanconi anemia-associated nuclease 1 (FAN1), that promotes ICL repair in a manner strictly dependent on its ability to accumulate at or near sites of DNA damage and that relies on mono-ubiquitylation of the ID complex. Thus, the mono-ubiquitylated ID complex recruits the downstream repair protein FAN1 and facilitates the repair of DNA interstrand cross-links.

SUMMARY Proteins that bind to single-stranded DNA (ssDNA) are essential for DNA replication, recombinational repair and maintenance of genomic stability. Here we describe the characterization of a ssDNA-binding heterotrimeric complex, SOSS (Sensor Of Single-stranded DNA) in human, which consists of human SSB homologues hSSB1/2 (SOSS-B1/2), INTS3 (SOSS-A) and a previously uncharacterized protein C9orf80 (SOSS-C). We have shown that SOSS-A serves as a central adaptor required not only for SOSS complex assembly and stability, but also for facilitating the accumulation of SOSS complex to DNA ends. Moreover, SOSS-depleted cells display increased IR sensitivity, defective G2/M checkpoint and impaired HR repair. Thus, our study defines a pathway involving the sensing of ssDNA by SOSS complex and suggests that this SOSS complex is likely involved in the maintenance of genome stability.

Mutations in HepA-related protein (HARP) are the only identified causes of Schimke immunoosseous dysplasia (SIOD). HARP has a unique annealing helicase activity in vitro, but the in vivo functional significance remains unknown. Here, we demonstrated that HARP is recruited to stalled replication forks via its direct interaction with Replication protein A (RPA). Cells with HARP depletion displayed increased spontaneous DNA damage and G2/M arrest, suggesting that HARP normally acts to stabilize stalled replication forks. Our data place the annealing helicase activity of HARP at replication forks and propose that SIOD syndrome may be caused by the destabilization of replication forks during cell proliferation. HARP (HepA-related protein), also known as SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), is a member of the SWI/SNF family of proteins (Eisen et al. 1995;Coleman et al. 2000;Flaus et al. 2006). Mutations in HARP are responsible for an autosomal recessive disorder known as Schimke immunoosseous dysplasia (SIOD), with the diagnostic features of spondyloepiphyseal dysplasia, renal dysfunction, and T-cell immunodeficiency (Schimke et al. 1971;Spranger et al. 1991;Boerkoel et al. 2000). Recent studies suggested that HARP has an unusual biochemical activity as an annealing helicase, which is opposite to helicases that are normally involved in DNA unwinding (Yusufzai and Kadonaga 2008). However, the exact biological function of this HARP annealing helicase activity remains unknown.In this study, we report that HARP associates with Replication protein A (RPA) in vitro and in vivo. RPA was initially identified as an ssDNA-binding protein that is absolutely required for DNA replication of simian virus 40 (SV40) (for reviews, see Waga and Stillman 1998;Stenlund 2003;Fanning et al. 2006). Human RPA is a stable heterotrimer composed of three subunits-RPA70, RPA32, and RPA14 (also named as RPA1, RPA2, and RPA3)-that are conserved among eukaryotes. RPA is essential for DNA replication, repair, and recombination, and DNA damage signaling pathways in eukaryotic cells (Zou and Elledge 2003;Binz et al. 2004;Stauffer and Chazin 2004b;Fanning et al. 2006). The functions of RPA in these diverse processes depend on its ssDNA-binding activity and its ability to interact with multiple proteins involved in these pathways (for reviews, see Fanning et al. 2006;Zou et al. 2006). Therefore, RPA can be considered as an adaptor protein that facilitates various biochemical reactions that occur at or involve ssDNA. The interaction between HARP and RPA suggest that HARP may function during replication and/or DNA repair. Indeed, our subsequent studies indicate that HARP is involved in the protection of stalled replication forks and thus provide a plausible mechanism for the development of SIOD syndrome. Results and DiscussionIn an effort to identify new RPA-associated proteins, we performed tandem affinity purification (TAP) using soluble or chromatin fraction prepared from 293T cells ...

Background: Translesion synthesis involves proliferating cell nuclear antigen (PCNA) monoubiquitination and polymerase switching. Results: C1orf124 is required for cell survival following UV damage. It binds to monoubiquitinated PCNA and participates in polymerase switching. Conclusion: C1orf124 serves as a central platform that facilitates translesion synthesis. Significance: This study provides a mechanism for translesion synthesis.

Summary Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in vitro and its function in vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrate the in vivo functions of AH2, but also reveal a common feature of this new subfamily of proteins with annealing activity.