New Players in the BRCA1-mediated DNA Damage Responsive Pathway

Introduction

External and internal hazards including reactive oxygen species, ionizing radiation (IR) and many chemicals can induce DNA double strand breaks (DSBs). In response to DSBs or stalled replication forks, various cell cycle checkpoints are activated. These checkpoints are believed to coordinate cell cycle progression with DNA repair or induce apoptosis when DNA damage cannot be repaired promptly. Therefore, DNA damage checkpoints are considered important self-defense mechanisms that ensure genomic stability (Rouse and Jackson, 2002; Su, 2006; Zhou and Elledge, 2000).

Many proteins including the protein kinase ataxiatelangiectasia mutated (ATM), γH2AX, mediator of DNA damage checkpoint protein 1 (MDC1), Nijmegen breakage syndrome 1 (NBS1), the product of Breast Cancer Susceptibility Gene 1 (BRCA1), and checkpoint kinases 1 and 2 (Chk1 and Chk2) are involved in the ionizing radiation (IR)-induced DNA damage response pathway (Su, 2006). Dysregulation of this pathway by mutations or deletions of genes involved contributes to genomic instability, which eventually leads to neoplastic transformation (Hahn and Weinberg, 2002). The proteins that participate in DNA damage response can be loosely divided into three groups. One is the so-called DNA damage sensors or upstream regulators, which are involved in the detection of DNA damage and the initiation of DNA damagesignaling cascade. ATM and ATR kinases can be included in this group. The second group is the mediators of the DNA damage response, which transfer the DNA damage signals from sensors to effecters. MDC1, 53BP1, TopBP1 and BRCA1 are included in this group. Most proteins in this group lack catalytic activity but facilitate signal transduction by mediating protein-protein interactions. The final one is the effectors, which includes Chk1 and Chk2. BRCA1 is the one of the mediator proteins involved in DNA damage signaling. Germline mutations in BRCA1 gene have been detected in approximately half of familial breast cancer cases and most cases of combined familial breast/ovarian cancers (Couch et al., 1997; Ford et al., 1998). BRCA1 mutant cell lines have defects in DNA damage response pathway and increased genomic instability (Deng, 2002; Deng and Brodie, 2000; Scully and Livingston, 2000; Venkitaraman, 2002), suggesting that BRCA1 is likely to function as care-taker tumor suppressor in vivo. The BRCA1 gene encodes all 1863 amino acid protein, which contains two specific regions, the N-terminal Ring domain that has E3 ligase activity and the tandem Cterminal BRCT motifs that specifically recognize phosphoserine/ theronine motifs (Chen et al., 2002; Hashizume et al., 2001; Manke et al., 2003; Yu et al., 2003). The C-terminal BRCT motifs, which have also been found in many proteins involved in the DNA damage response pathway, are very important for the localization of BRCA1 to DNA damage sites and BRCA1 function in transducing DNA damage signals.

BRCA1-associated C-terminal Helicase (BACH1) and C-terminal binding protein-interacting protein (CtIP) are two known BRCA1 BRCT domain-binding partners that associate with BRCA1 in a phosphorylation-dependent manner (Cantor et al., 2001; Yu and Chen, 2004). CtIP transiently binds to BRCA1 in late S or G2 phase and is involved in CHK1 phosphorylation and G2/M checkpoint control, while the interaction between BACH1 and BRCA1 starts in S phase and is required for the damage-induced Sto- G2 transition (Yu and Chen, 2004). These two BRCA1- associated proteins are believed to work downstream of BRCA1 in response to DNA damage. Recently, several proteins that operate upstream of BRCA1 have been identified. These include the Ring domain nuclear factor 8 (RNF8) (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007), receptor associated protein 80 (RAP80) (Kim et al., 2007a; Sobhian et al., 2007; Wang et al., 2007) and coiled-coil domain containing protein 98 (CCDC98) (Kim et al., 2007b; Liu et al., 2007; Wang et al., 2007). These proteins participate in the recruitment of BRCA1 to DNA damage sites and function with BRCA1 in G2/M checkpoint control.

In this review, we will discuss the mechanism by which BRCA1 is recruited to the sites of DNA damage and how BRCA1 mediates the transduction of DNA damage signals to downstream effectors.

Receptor associated protein 80 (RAP80)

RAP80 was originally identified as a retinoid-related testis associated protein using yeast two hybrid system (Yan et al., 2002). The RAP80 gene encodes a protein of 719 amino acids, which contains tandem ubiquitin-interact-ing motifs (UIM) domain at its N-terminus and tandem zinc finger domains at its C-terminus. More recently, several groups identified RAP80 as a BRCA1-associated protein that functions in DNA damage response (Kim et al., 2007a; Sobhian et al., 2007; Wang et al., 2007). Upon DNA damage, RAP80 re-localizes to the sites of DNA breaks and is phosphorylated at several residues in an ATM-dependent manner (Kim et al., 2007a; Sobhian et al., 2007; Wang et al., 2007). Indeed, ATM deficient (FT169A) cell lines show a severe defect in RAP80 phosphorylation following ionizing radiation, indicating that ATM may be the primary kinase for RAP80 phosphorylation in response to DSBs. But, the importance of RAP80 phosphorylation in DNA damage response remains to be clarified.

RAP80 contains both Ubiquitin interacting motifs and zinc finger domains. While the function of the putative zinc finger domain of RAP80 is unclear, RAP80 UIMs, which are found in many proteins including HSJ1A, 26S proteosome subunit S3a, USP25, USP28, HRS, STAM and Epsin1, 2, and 3, plays an important role in targeting RAP80-CCDC98/Abraxas-BRCA1 complex to the DNA damage sites and is required for the ionizing radiationinduced foci (IRIF) formation (Kim et al., 2007a; Sobhian et al., 2007; Wang et al., 2007), leading to the idea that RAP80 localizes at the DNA damage sites through binding with unknown ubiquitinated protein(s). Interestingly, several studies showed that the RAP80 UIMs bind specifically to lysine 63-linked and lysine 6-linked polyubiquitin chains, but not lysine 48-linked polyubiquitin chains in vivo and in vitro (Kim et al., 2007a; Sobhian et al., 2007). In support of protein ubiquitination in RAP80 recruitment, lysine 6-linked and lysine 63-linked ubiquitin polymers were observed in cells following DNA damage (Kim et al., 2007a; Sobhian et al., 2007). These ubiquitin chains colocalize with BRCA1 at DNA damage sites and the localization of these ubiquitin chains at DNA damage sites depends on the upstream mediator MDC1 (Kim et al., 2007a; Sobhian et al., 2007). Moreover, point mutations in RAP80 UIM regions diminish the association of these mutants with ubiquitin in vitro and decrease the damage-induced RAP80 foci formation in vivo (Kim et al., 2007a). Together, these data suggest that RAP80 is targeted to DNA damage sites through binding to MDC1-dependent lysine 63-linked and/ or lysine 6-linked ubiquitin polymers at the sites of DNA breaks.

Coiled-coil domain-containing protein 98

(CCDC98)/Abraxas CCDC98/Abraxas was identified as a protein that interacts with both RAP80 and BRCA1 (Kim et al., 2007b; Liu et al., 2007; Wang et al., 2007). CCDC98/Abraxas contains a central coiled-coil domain and a 406SPTF409 motif at its extreme C-terminus. This SPTF motif of CCDC98/Abraxas is identical to the phosphorylated sequence in BACH1 that is required for its interactiom with BRCA1 BRCT domains (Yu and Chen, 2004). Indeed, the serine residue in the SPTF motif of CCDC98/Abraxas is phosphorylated and required for the phosphorylationdependent interaction between CCDC98/Abraxas and BRCA1 (Kim et al., 2007b; Liu et al., 2007; Wang et al., 2007).

CCDC98/Abraxas rapidly translocates to DSB sites via its N-terminal region (residues 1–250), which is the same region required for its association with RAP80. The CCDC98/Abraxas-binding domain on RAP80 is mapped to a region (residues 235–337) that localizes C-terminal to the UIMs of RAP80. Since the same region of RAP80 (residues 235–337) is also critical for its association with BRCA1 in vivo, these observations suggest that CCDC98 may serve as a bridging protein between RAP80 and BRCA1. In support of this hypothesis, BRCA1 did not accumulate at the sites of DNA breaks in CCDC98 knockdown cells, while the relocalization of RAP80 to damage sites was normal. Moreover, both BRCA1 and CCDC98 foci formation were abolished in RAP80 depleted cells, but the focus localization of CCDC98 and RAP80 was normal in BRCA1 depleted cells (Kim et al., 2007b; Liu et al., 2007; Wang et al., 2007). Collectively, these data indicate that CCDC98 acts downstream of RAP80 but upstream of BRCA1 in response to DNA damage.

Ring domain nuclear factor 8 (RNF8)

RNF8 was first identified as a RING domain-containing protein that interacts with class III human ubiquitinconjugating enzymes (E2s) (Ito et al., 2001). RNF8 was shown latter to interact with Retinoid X receptor and regulate its transcriptional activation (Takano et al., 2004). The RNF8 gene encodes a protein of 485 residues, which contains a Forkhead-associated (FHA) domain at its N-terminus and a RING domain at its C-terminus. Recently, studies from several groups suggest that RNF8 is a key mediator involved in the assembly of multiple checkpoint proteins to the sites of DNA breaks (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007).

We and others have found that RNF8 FHA domain prefers to bind to pTxxY/F phosphomotifs in vitro (Huen et al., 2007; Mailand et al., 2007). In vivo, the FHA domain of RNF8 is required for the ionizing radiation-induced foci (IRIF) formation of RNF8 (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007), leading to the idea that RNF8 localizes to the DNA damage sites through binding with phosphorylated proteins at DNA damage sites. Indeed, the RNF8 FHA domain binds to the several conserved pTQXF motifs (T699QCF, T719QAF, T752QPF and T765QPF) within MDC1. In addition, RNF8 IRIF formation was abolished in MDC1-deficient cells, and wild-type MDC1, but not MDC1 mutants defective in RNF8 binding, could restore RNF8 IRIF in MDC1-deficient cells (Huen et al., 2007; Mailand et al., 2007). These data suggest that MDC1 mediates the translocation of RNF8 to DNA damage sites via a phosphorylation-dependent interaction between these conserved phosphorylated motifs on MDC1 and the FHA domain of RNF8.

Besides the N-terminal FHA domain, RNF8 also has a RING domain at the C-terminus that possesses E3 ubiquitin ligase activity. While this domain is not required for its translocation to DNA damage sites, it is critical for the subsequent recruitment of the RAP80/Abraxa, BRCA1 and 53BP1 to the sites of DNA damage (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007), indicating that ubiquitination of some components at or near the sites of DNA breaks by RNF8 is a prerequisite for the IRIF formation of these downstream checkpoint proteins.

The potential ligase function of RNF8 is further supported by the fact that the phenotypes observed in RNF8 depleted cells are very similar to those in cells with Ubc13 deficiency (Zhao et al., 2007). Ubc13 is the only known E2 conjugating enzyme that can catalyze the formation of Lys63-linked polyubiqiuin chains. Like RNF8, Ubc13 is important for the formation of ubiquitin conjugates and the recruitment of BRCA1 and 53BP1 following DNA damage (Zhao et al., 2007). Therefore we speculate that RNF8 may function together with Ubc13 at the sites of DNA breaks and promote protein ubiquitination and checkpoint protein assembly at DSBs. Given the importance of RNF8 in DNA damage signaling transduction, it is not surprising that the G2/M checkpoint defect and reduced cell survival following radiation were observed in cells with RNF8 deficiency (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007).

A current model of signal transduction network activated following DNA damage

Based on the data presented in this review, we propose the following model for many protein’s functions in DNA damage response: following DNA damage (Fig. 1). The key upstream sensor initiates the DNA damage response is the large protein kinase ATM, which is activated and autophosphorylated at several residues including Ser 1981 in response to DNA double stranded breaks. Immediately following ATM activation, ATM can phosphorylate a histone H2A variant H2AX at residue Ser 139 at or near the sites of DNA breaks. Because H2AX is phosphorylated right next to the sites of DNA breaks, this phosphorylated form of H2AX (named γ-H2AX) has been widely used as a marker of DNA double stranded breaks. This phosphorylated H2AX can bind directly to MDC1 BRCT domains and thus accumulate MDC1 to DNA damage sites (Lou et al., 2006; Stucki et al., 2005). At or near the sites of DNA damage, MDC1 can directly or indirectly (via Mre11/NBS1/ Rad50 complex) recruit additional active ATM to the sites of DNA damage (Falck et al., 2005; Lou et al., 2006). This accumulation of active ATMs at DSBs should result in the further phosphorylation of H2AX at mega-base region surrounding any single DNA break and provide docking sites not only for MDC1, but also other checkpoint proteins at or near the sites of DNA breaks. We believe that this positive feedback mechanism amplifies the DNA damage signals and is especially critical for checkpoint activation following low doses of DNA damage.

Early studies suggest that H2AX and MDC1 are also required for the accumulation of many downstream checkpoint proteins. These include Mre11/Rad50/Nbs1 protein complex, RNF8, 53BP1 and RAP80/CCDC98/BRCA1 complex at the sites of the DNA breaks. It is likely that a direct interaction between MDC1 and Mre11/Rad50/Nbs1 complex is involved in the recruitment of Mre11/Rad50/Nbs1 complex (Bekker-Jensen et al., 2005; Stucki et al., 2005). Similarly, recently studies support that a direct interaction between MDC1 and RNF8 is required for localizing RNF8 to the sites of DNA breaks (Huen et al., 2007; Mailand et al., 2007). RNF8 is the key linker molecule, which facilitates the transduction of DNA damage signals from H2AX/MDC1 to downstream checkpoint and repair proteins including 53BP1, RAP80/CCDC98 and BRCA1 (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007). We now know precisely how this signal transduction event is regulated in the cell. RNF8 utilizes its FHA domain to bind directly to phosphorylated MDC1 following DNA damage and thus is specifically recruited only after DNA damage. Once at the sites of DNA breaks, RNF8 RING domain, together with the E2 ubiquitin conjugase Ubc13, ubiqutinates several substrates including H2AX and H2A/H2B at or near the sits of DNA damage. The ubiquitination of these RNF8 substrates create docking sites for the UIM domain of RAP80 and therefore recruit the RAP80/CCDC98 and subsequently BRCA1 complex to DNA damage sites. BRCA1 at sites of DNA damage not only is phosphorylated by ATM and ATR, but also binds to BACH1 and CtIP, ubiquitinates CtIP and activates downstream checkpoint kinase CHK1. Therefore BRCA1 functions as a mediator in the DNA damage response pathway.

Summary

Recent studies have revealed that in addition to protein phosphorylation, protein ubiquitination also plays important roles in the DNA damage response pathway. Coordination of these posttranslational modifications provides the complexity that allows for fine-tuning of the DNA damage-signaling network. It will be exciting to further define how protein ubiquitination regulates DNA damage checkpoints and DNA repair in this multifaceted signaling network.