The role of BRCA1 in DNA damage response

Abstract

BRCA1 is a well-established tumor suppressor gene, which is frequently mutated in familial breast and ovarian cancers. The gene product of BRCA1 functions in a number of cellular pathways that maintain genomic stability, including DNA damage-induced cell cycle checkpoint activation, DNA damage repair, protein ubiquitination, chromatin remodeling, as well as transcriptional regulation and apoptosis. In this review, we discuss recent advances regarding our understanding of the role of BRCA1 in tumor suppression and DNA damage response, including DNA damage-induced cell cycle checkpoint activation and DNA damage repair.

Keywords

BRCA1 / DNA damage response / tumorigenesis

INTRODUCTION

Breast cancer is one of the most common cancers in women, accounting for over 20% of all cancer cases. Among them, 5%–10% of breast cancer cases are ascribed to hereditary predisposition (Alberg and Helzlsouer, 1997). Using linkage analysis, BRCA1 was identified as the first breast cancer susceptibility gene (Hall et al., 1990). Germline mutations of BRCA1 have been found to predispose women to high risk of breast and ovarian cancers (Futreal et al., 1994; Brody and Biesecker, 1998; Venkitaraman, 2002). BRCA1 mutations in germline usually occur in one allele, while the other healthy allele is further mutated or lost during cancer development.

BRCA1 gene contains 24 exons that encode an 1863-residue nuclear protein in human (Miki et al., 1994). The exon 11 is the largest exon and encodes over 60% of amino acids of BRCA1. Although it shares very limited homology with other known proteins, BRCA1 contains two functional domains: an N-terminal Ring domain and a C-terminal BRCT domain. Similar to other Ring domains, the Ring domain of BRCA1 has E3 ubiquitin ligase activity and facilitates protein ubiquitination (Panier and Durocher, 2009). The C-terminal BRCT domain of BRCA1 is a phospho-protein binding domain (Manke et al., 2003; Rodriguez et al., 2003; Yu et al., 2003), which is also required for BRCA1’s translocation and accumulation at DNA damage sites (discussed later). Although mutations in BRCA1 are scattered throughout the gene body, many cancer-associated mutations have been found within the Ring domain and the BRCT domain of BRCA1, indicating that both domains are very important to suppress breast and ovarian cancer formation (Monteiro et al., 1996; Brzovic et al., 2001; Williams and Glover, 2003).

Since the discovery of BRCA1 gene, several genetically engineered mouse models have been created for studying the role of BRCA1 in vivo. Until now, at least nine different conventional BRCA1 knockout mouse strains have been generated and characterized. Each strain carries a mutation in different part of BRCA1 to generate null allele (Evers and Jonkers, 2006; Bouwman and Jonkers, 2008). Deletion of BRCA1 in mice was embryonic lethal, which was accompanied by growth retardation, apoptosis, cell cycle defects and genomic instability, suggesting that BRCA1 is essential for early embryonic development (Deng, 2002). Unlike that in human, the spontaneous tumor penetrance in BRCA1^+/- mice was similar to that in wild-type mice, mainly because the wild-type BRCA1 allele was rarely affected during the short life span of mouse. However, with low-dose ionizing radiation (IR) treatment, BRCA1 heterozygous mice were prone to ovarian cancer, suggesting that this animal model could be very useful to analyze the mechanism of ovarian tumorigenesis (Jeng et al., 2007). In addition, conditional BRCA1 knockout mice have been generated by Cre recombinase-mediated deletion of genomic regions flanked by loxP recombination site in special tissues. Up to date, at least five different conditional BRCA1 knockout mouse strains have been generated (Xu et al., 1999; Mak et al., 2000; Liu et al., 2007a; McCarthy et al., 2007; Shakya et al., 2008). However, mammary tumorigenesis occurred at low frequency after long latency in these conditional knockout mice, suggesting that other genetic hits may cooperate with loss of BRCA1 together to induce breast neoplasia (Liu et al., 2007a). Indeed, genetic deletion of both BRCA1 and p53 significantly accelerated breast cancer formation, suggesting the genetic interaction between BRCA1 and p53 in tumor suppression (Liu et al., 2007a).

Although the precise role of BRCA1 in breast tumor prevention remains elusive, accumulating evidence suggests that BRCA1 could be one of the key players in DNA damage response. Double-stranded DNA inside cell nucleus constantly encounters damages induced by both external and internal hazards, such as IR, UV and oxidative stress. If not correctly treated, these damages will be accumulated along with DNA replication and be passed into daughter cells. Accumulated DNA damage will cause genomic instability and finally lead to tumorigenesis. In the presence of BRCA1, cells could sense and repair DNA lesions, which ensures genomic integrity and prevents tumorigenesis, whereas cancer-associated BRCA1 mutations disrupt normal DNA damage response. Therefore, the pivotal roles of BRCA1 in DNA damage response might explain itself as an important tumor repressor. Here, we will discuss the role of BRCA1 in DNA damage response, including the molecular mechanisms by which BRCA1 is recruited to DNA damage sites and by which BRCA1 promotes DNA damage checkpoint activation and DNA damage repair.

BRCA1 IS RECRUITED TO DNA DAMAGE SITES

The most direct and obvious evidence supporting BRCA1’s roles in DNA damage response is that BRCA1 relocates to DNA damage sites and forms nuclear foci following DNA double-strand breaks (DSBs) (Scully et al., 1997a). Although this phenomenon was observed more than 10 years ago, the signaling cascade that triggers BRCA1’s translocation remains largely unknown until recently. Following DNA damage, chromatin-associated histone H2AX that locates close to DNA damage sites is phosphorylated by ATM and ATR (Burma et al., 2001), and subsequently recruits a phospho-module binding mediator MDC1 and an E3 ubiquitin ligase RNF8 to DNA damage sites (Stucki et al., 2005; Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007). RNF8 functions together with an E3 ubiquitin conjugase Ubc13 to ubiquitinate histone H2A and H2B at chromatin lesions, which regulates the translocation of BRCA1 to DNA damage sites (Zhao et al., 2007; Wu et al., 2009). Using protein affinity purification approaches, we and others have identified a novel BRCA1 complex recently, including BRCA1, RAP80, CCDC98/Abraxas, NBA1/MERIT40, BRCC36 and BRCC45 (Kim et al., 2007a, b; Liu et al., 2007b; Sobhian et al., 2007; Wang et al., 2007; Feng et al., 2009; Shao et al., 2009; Wang et al., 2009). Following DNA damage, RAP80 recognizes ubiqutinated histone at the site of DNA damage via its ubiquitin-interacting motif (UIM) and recruits the big complex, including BRCA1 to DNA damage sites (Wu et al., 2009).

BRCA1 IS IMPORTANT FOR DNA DAMAGE-INDUCED CELL CYCLE CHECKPOINTS ACTIVATION

Cell cycle checkpoints serve to monitor the chromatin status during cell cycle, which ensures that cell cycle proceeding normally (Hartwell and Weinert, 1989). Upon DNA damage, cell cycle checkpoints are activated to arrest cells at certain stage during cell cycle, which allow cells to have enough time to repair damaged DNA before resuming cell cycle progression (Hartwell and Kastan, 1994). The checkpoint activation following DNA damage is critical for maintaining genomic integrity as it guards against duplication of damaged DNA and passage of damaged DNA to daughter cells. Consistently, dysfunction of proteins involved in cell cycle checkpoints often results in developmental abnormality, genomic instability and tumorigenesis (Lobrich and Jeggo, 2007). According to the different stages during cell cycle where they function, cell cycle checkpoints can be categorized into G1/S, S-phase and G2/M checkpoints, and BRCA1 has been suggested to be responsible for all of these checkpoints activations.

G1/S checkpoint

Following DNA damage, the G1/S checkpoint arrests cells at G1/S boundary, which prevents damaged DNA in G1 cells to be used for upcoming DNA replication. The tumor suppressor p53 plays a critical role in DNA damage-induced G1/S checkpoint by controlling cyclin inhibitor p21’s transcription (Kuerbitz et al., 1992; Dulic et al., 1994; Reed et al., 1994). Using siRNA to downregulate BRCA1, Fabbro et al showed that BRCA1-depleted cells had defective G1/S checkpoint in response to DNA damage (Fabbro et al., 2004). They demonstrated that BRCA1 acted as a scaffold protein facilitating phosphorylation of p53 by ATM in response to DNA damage, which led to p53-mediated induction of p21 and induced G1/S arrest. In this study, they also demonstrated that although BRCA1 was required for both IR and UV-induced p53 phosphorylation by ATM/ATR, BRCA1 was only required for IR-induced G1/S checkpoint, but not for UV-induced G1/S arrest (Fabbro et al., 2004). However, a recent study indicated that primary fibroblasts from human BRCA1 heterozygotes displayed a moderate impaired G1/S cell cycle checkpoint compared with wild-type cells after UV treatment (Shorrocks et al., 2004). Nevertheless, BRCA1 mediating ATM-dependent p53 phosphorylation demonstrates the functional interaction between these two important tumor suppressors in the G1/S checkpoint.

S-phase checkpoint

Another cell cycle checkpoint induced by DNA damage is S-phase checkpoint, which suppresses S phase progression and stops DNA replication immediately following DNA damage (Larner et al., 1997). Since S-phase checkpoint is defective in BRCA1-deficient HCC1937 cells and complementation of functional BRCA1 in this cell line can restore the S-phase checkpoint following DNA damage, it demonstrates that BRCA1 plays an essential role in S-phase checkpoint activation (Xu et al., 2001). Like BRCA1, deficiency of many DNA damage response proteins, such as ATM, ATR, Chk1 and Chk2, has been shown to cause defects in DNA damage-induced S phase checkpoint (Painter, 1981; Lim et al., 2000; Falck et al., 2001; Yazdi et al., 2002). Following DNA damage, ATM and ATR are activated and stimulate Chk1 and Chk2. Activated Chk1 and Chk2 regulate a family of phosphates Cdc25 A/B/C, which governs cyclins and cyclin-dependent kinases’ activity during S phase progression (Zhao et al., 2002). Although the detail mechanism is not clear, BRCA1 participates in this signal transduction by regulating Chk1 kinase activity (Yarden et al., 2002). Moreover, ATM-mediated Ser1387 phosphorylation of BRCA1 is specifically required for the S-phase checkpoint following IR, suggesting that phospho-BRCA1 may recruit functional partners for regulating this signal cascade (Xu et al., 2001). In addition, BRCA1 may also regulate ATM activation following DNA damage in S phase. It has been shown that BRCA1 interacted and colocalized with Mre11/Rad50/NBS1 (MRN) complex (Zhong et al., 1999; Wu et al., 2000a), which is a sensor for DSBs and directly activates ATM (Lee and Paull, 2004, 2005).

G2/M checkpoint

Besides G1/S and S-phase checkpoint, DNA damage also activates G2/M checkpoint, which transiently arrest cells at G2/M boundary. It allows cells to repair DNA lesions prior to mitosis and prevents damaged DNA being passed to daughter cells. The mechanism of G2/M checkpoint has been well studied. Upon DNA damage, Chk1 and Chk2 are phosphorylated and activated by ATM and ATR. Activated Chk1 and Chk2 then phosphorylate mitotic kinase Weel and Cdc25A/B/C, which suppress the activity of cyclin B and Cdc2 and block cells entering mitosis (O'Connell et al., 1997; Rhind et al., 1997; O'Connell et al., 2000; Cuddihy and O'Connell, 2003). Loss of BRCA1 abolishes this G2/M checkpoint action. Like in S-phase checkpoint activation, BRCA1 regulates Chk1 kinase activity during G2/M checkpoint activation (Yu and Chen, 2004). Distinct from in S-phase checkpoint activation, ATM phosphorylates Ser1423 of BRCA1, which is required for G2/M checkpoint activation, suggesting that BRCA1 may have different functional partners to mediate G2/M checkpoint activation analogous to S-phase checkpoint activation (Xu et al., 2001).

BRCA1 PROMOTES DNA DAMAGE REPAIR

BRCA1 was first implicated in DNA damage repair because it translocated to DNA damage sites and colocalized with RAD51, an essential protein in homologous recombination repair (Scully et al., 1996, 1997b). Later on, many studies from different groups have demonstrated that BRCA1-deficient cells were hypersensitive to DNA damage agents such as IR, UV and DNA alkylating agents and impaired DNA damage repair, further suggesting that BRCA1 plays an important role in DNA repair (Scully et al., 1999).

In response to different types of DNA damage, different DNA repair processes utilize different repair machineries, including homologous recombination (HR), non-homologous end-joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR) (Murakami and Kawasaki, 1975; Jeggo, 1998; Dasika et al., 1999; Harfe and Jinks-Robertson, 2000; Bernstein et al., 2002). Among them, NHEJ and HR are two predominant repair pathways for DSBs, the most deleterious damage on the chromatin. Furthermore, BRCA1 participates in both types of DNA repair.

BRCA1 and NHEJ

NHEJ is the most common form of DNA repair in cells, which mainly occurs during G1 phase. Unlike HR, which faithfully repairs damaged DNA, NHEJ is a relatively error-prone type of repair without using additional template. The DNA damage ends with limited processing are directly ligated, which results in removal or addition of bases at broken ends. BRCA1 has been implicated in the NHEJ repair pathway, although this remains contentious with numerous studies presenting conflicting results using both in vivo and in vitro assays. The most possible explanation for BRCA1 being involved in NHEJ is that BRCA binds to MRN complex both in vitro and in vivo, although the mechanism of this interaction is still unclear (Fu et al., 2003). MRN complex plays an major role in both NHEJ and HR repair (Fu et al., 2003). There is evidence that BRCA1 can suppress the nuclease activity of MRE11 and BRCA1 is required for ATM-dependent phosphorylation of NBS1 following DNA damage (Zhong et al., 1999; Wang et al., 2000; Wu et al., 2000b; Paull et al., 2001; Foray et al., 2003). In addition, many studies have provided evidence that the NHEJ pathway was defective in BRCA1-deficient MEFs, human BRCA1-deficient cells HCC1937 and lymphoblastoid cells obtained from women carrying truncation or missense BRCA1 mutations using different assays (Baldeyron et al., 2002; Zhong et al., 2002; Bau et al., 2004; Coupier et al., 2004; Bau et al., 2006). However, these assays are either indirect or nonspecific for NHEJ. Meanwhile, previous studies have also shown conflicting results on the role of BRCA1 in NHEJ. It has been shown that BRCA1-deficient mouse ES cells were proficient in NHEJ repair and the random plasmid integration rate of BRCA1-deficient mouse ES cells was higher than that of control wild type cells (Moynahan et al., 1999; Snouwaert et al., 1999). Moreover, pulse-field gel electrophoresis displays similar DSB repair kinetics in HCC1937 and wild type BRCA1-complemented HCC1937 cells, suggesting that BRCA1 is not required for NHEJ (Wang et al., 2001). To understand and explain why these observations are inconsistent, it is essential to understand the mechanisms of NHEJ repair and each assays used to detect NHEJ, as different assays may examine different repair mechanisms.

BRCA1 and HR

Unlike NHEJ, HR occurs only during S and G2 phase of the cell cycle when sister chromatids are present. HR is activated by DNA damage and relies on ATM and MRN complex-mediated resection of double-stranded broken ends into single-stranded DNA (ssDNA). Then ssDNA are coated by RPA, a group of ssDNA binding proteins, forming substrates for loading the recombinase RAD51, which catalyses invasion of ssDNA into sister chromatid. Using sister chromatid as the template, ssDNA is elongated and holiday junctions are formed between two sister chromatids. The holiday junction is then resolved and DNA ends are ligated in an error-free manner (West, 2003). BRCA1 interacts with MRN complex during HR repair, indicating that BRCA1 may participate in MRN-dependent DNA end resection (Greenberg et al., 2006). In addition, recently, we and others identified that BRCA1 interacted with PALB2 and BRCA2 at DNA damage sites (Sy et al., 2009; Zhang et al., 2009a, b). Both PALB2 and BRCA2 are functional partners of RAD51 and facilitate RAD51-ssDNA filament formation (Xia et al., 2006). Loss of BRCA1 disrupts the stability of PALB2 and BRCA2 at DNA damage sites, which in turn abolishes RAD51’s localization at DNA lesions and abrogates HR repair (Zhang et al., 2009a, b).

Recently, accumulated evidence shows that BRCA1 and BRCA2-deficient cells are hypersensitive to Poly(ADP-ribose) polymerase (PARP) inhibitors, suggesting that PARP inhibitors can be employed as novel therapeutic drugs to selectively treat BRCA1 or BRCA2-deficient breast tumors (Bryant et al., 2005; Farmer et al., 2005). PARP is a key regulator in base excision repair process and participates in repair of DNA single strand breaks (SSBs) (Bouchard et al., 2003). Loss of PARP activity is likely to cause the accumulation of SSBs, which are converted to DSBs during replication or HR repair (Curtin, 2005). The increased DNA lesions result in the lethality of BRCA1 or BRCA2-deficient cells. PARP inhibitors in the recent clinical trials have shown profound antitumor activities in breast, ovarian and prostate cancers with BRCA1 or BRCA2 mutations (Fong et al., 2009). Thus, PARP1 inhibitors are likely to be promising drugs for clinical treatment of BRCA1 and BRCA2-deficient tumors (Bolderson et al., 2009).

CONCLUSIONS

We have examined evidence supporting BRCA1’s important role in DNA damage response, including cell cycle checkpoint activation and DNA damage repair. Although the molecular mechanisms underlying BRCA1’s roles in DNA damage response are emerging, they are far from clear, and many discrepancies still exist. Insights into the mechanisms of BRCA1 in checkpoint regulation and DNA damage repair will help us understand the molecular mechanisms by which BRCA1 maintains the genomic stability and contributes to tumor suppression in vivo, and ultimately find effective ways to prevent breast cancer development.

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