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Cancer Lett. Author manuscript; available in PMC 2009 Nov 28.
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PMCID: PMC2749678
NIHMSID: NIHMS78340

RAP80 and RNF8, key players in the recruitment of repair proteins to DNA damage sites

Abstract

Chromosomal double-strand breaks (DSBs) in eukaryotes provoke a rapid, extensive modification in chromatin flanking the breaks. The DNA damage response (DDR) coordinates activation of cell cycle checkpoints, apoptosis, and DNA repair networks, to ensure accurate repair and genomic integrity. The checkpoint kinase ATM plays a critical role in the initiation of DDR in response to DSBs. The early ATM-mediated phosphorylation of the histone variant H2AX proteins near DSBs leads to the subsequent binding of MDC1, which functions as a scaffold for the recruitment and assembly of many DDR mediators and effectors, including BRCA1. Recent studies have provided new insights into the mechanism by which BRCA1 and associated proteins are recruited to DNA damage foci and revealed key roles for the receptor-associated protein 80 (RAP80) and the E3 ligase RNF8 in this process. RAP80 is an ubiquitin-interaction motif (UIM) containing protein that is associated with a BRCA1/BARD1 complex through its interaction with CCDC98 (Abraxas). The UIMs of RAP80 are critical for targeting this protein complex to DSB sites. Additional studies revealed that after binding γ-H2AX, ATM-phosphorylated MDC1 is recognized by the FHA domain of RNF8, which subsequently binds the E2 conjugating enzyme UBC13. This complex catalyzes K63-linked polyubiquitination of histones H2A and γH2AX, which are then recognized by the UIMs of RAP80, thereby facilitating the recruitment of the BRCA1/BARD1/CCDC98/RAP80 protein complex to DSB sites. Depletion of RAP80 or RNF8 impairs the translocation of BRCA1 to DNA damage sites and results in defective cell cycle checkpoint control and DSB repair. In this review, we discuss this cascade of protein phosphorylation and ubiquitination and the role it plays in the control of cellular responses to genotoxic stress by regulating the interactions, localization, and function of DDR proteins.

Keywords: double strand breaks, RAP80, RNF8, ubiquitination, CCDC98/Abraxas, BRCA1, UIM, MDC1

1. Introduction

DSBs are highly toxic DNA lesions that frequently develop endogenously from reactive oxygen species generated by cellular metabolism, by nucleases, and during DNA replication [1, 2]. They can also occur during T-cell receptor rearrangements in lymphocytes and during meiosis in germ cells [3-5]. DSBs can be induced exogenously by a variety of DNA damaging agents, such as ionizing radiation (IR), radiomimetic drugs, and environmental chemicals. To ensure genomic integrity, cells have developed an extensive DDR that senses DNA damage and catalyzes a multifaceted response that coordinates cell cycle arrest, apoptosis, and DNA repair networks [6-11]. The regulation of cell cycle checkpoints and DNA repair pathways are strictly coordinated and under tight control to guarantee accurate DSB repair and to prevent the introduction of errors in critical genes that may promote oncogenesis [12]. If not properly repaired, cells can undergo apoptosis or senescence. Dysregulation or impaired function of proteins involved in DDR can result in chromosomal changes, genome instability, and increase cancer risk [1, 2, 10, 11, 13].

Cells activate distinct signaling pathways in response to various types of DNA damages. Nonhomologous end joining (NHEJ) and homologous recombinational repair (HRR), the main DSB repair mechanisms, are part of such responses to DSBs. DDRs are mediated through a large number of proteins classified as sensors, transducers, mediators, and effectors that play a role at different steps of the DDR. One of the first steps in HRR is the recognition of the DNA damage by the multifunctional protein sensor complex MRN (MRE11-RAD50-Nijmegen breakage syndrome 1 (NSB1)) that consists of the structure-specific nuclease MRE11, the ATPase and adenylate kinase subunit RAD50, and the adaptor protein NSB1. MRN complexes can bind to the exposed DNA ends directly and unwind the DNA ends in an ATP-dependent manner [6-9, 14, 15]. After DNA damage, many DDR proteins are spatially reorganized and accumulate into subnuclear structures, referred to as ionizing radiation-induced foci (IRIF) that surround DNA damage sites. The recruitment and assembly of these proteins into IRIF occurs in a temporal manner suggesting that this process is hierarchical and highly organized. This re-localization, as well the activity and function of these proteins, are under stringent control of multiple post-translational modifications, including acetylation, phosphorylation, ubiquitination, and sumoylation. One of the earliest events in DDR is the recruitment of the phosphoinositide-3-kinase-related protein kinase (PIKK) ATM by the MRN sensor complex [16-19]. This leads to the dissociation of the inactive dimer, autophosphorylation, and activation of ATM. Phosphorylation of H2AX and the subsequent recruitment of MDC1 are early events in DDR and essential for the recruitment and assembly of many DDR complexes, including MRN and the breast cancer-associated suppressor BRCA1. Recently, great progress has been made in understanding the mechanism by which BRCA1 and associated proteins are recruited to DBSs [8, 20, 21]. In this paper, we provide an overview of the critical roles that CCDC98/Abraxas, RAP80, MDC1, RNF8, and UBC13 play in this process.

2. Early cellular responses to DSBs

2.1 Phosphorylation of H2AX

The recruitment of DNA damage signal mediators and repair proteins to chromatin flanking DSBs occurs in a highly coordinated manner and with different kinetics indicating that this process is hierarchical [6-9]. This also suggests that these proteins act at different steps of the DDR and perform different functions in the networks regulating cell cycle checkpoints and DNA repair. It has been generally accepted that ATM phosphorylation of the histone H2A variant, H2AX, is one of the initial signals required for the subsequent recruitment of many DDR proteins to sites of DNA damage [22, 23]. Phosphorylation of H2AX (referred to as γ-H2AX) on a conserved serine at its C-terminus occurs within seconds after the induction of DSBs [24] and is dependent on the activation and targeting of ATM to DSB sites by the MRN complex that involves direct binding of ATM to the C-terminus of NBS1 [16, 25]. After IR treatment γ-H2AX is confined to IRIF and co-localizes with foci formed by other DDR-related proteins. γ-H2AX, particularly its phosphorylated C-terminus, is of critical importance for the recruitment of DDR proteins to IRIF because many mediator and repair-related proteins fail to translocate to nuclear foci in H2AX-depleted cells when its ATM phosphorylation site is mutated [26, 27]. The critical role of H2AX in DDR is further indicated by studies showing that knockdown of H2AX in cells increases genomic instability [23] and that H2AX knockout mice are more sensitive to radiation and exhibit an increased risk of developing cancer compared to wild type animals [26]. Interestingly, although BRCA1, 53BP1, and NBS1 are recruited to DSB sites in H2AX-deficient MEFs after laser microirradiation, they fail to form IRIF subsequently [26, 27] indicating that γ-H2AX is not essential for the initial re-localization of repair proteins to damaged chromatin, but is required for the accumulation of these proteins (foci formation) near DNA lesions.

2.2 Damage signal amplification by MDC1

The critical function of the C-terminal phosphopeptide of H2AX in the recruitment of DDR factors to IRIF suggested that this motif might provide a binding recognition site for one or more DDR proteins. Many proteins associated with IRIF contain domains known to recognize phosphopeptides, including the BRCA1 carboxyl-terminal repeat (BRCT) and forkhead-associated (FHA) domains, and therefore would be suitable candidates for such interaction. Recent studies identified mediator of DNA damage checkpoint 1 (MDC1), which contains one FHA at its N-terminus and two BRCT motifs at its C-terminus, as the protein binding the C-terminal phosphopeptide of γ-H2AX through its BRCT domain [28, 29]. MDC1 is recruited to IRIF within seconds after DSB damage. The phenotype of MDC1 knockout mice exhibits many similarities with that of H2AX knockout mice [28]. Loss of MDC1 expression impairs several cellular responses to DSBs, including control of intra-S and G2/M checkpoints and IR-induced apoptosis [30, 31]. Studies have indicated that the binding of MDC1 to γ-H2AX serves multiple functions. MDC1 appears to shield the C-terminus of γ-H2AX from premature dephosphorylation thereby promoting completion of DNA repair [29, 30]. In addition, MDC1 has been proposed to function in the amplification of the DNA damage signal [28, 32]. MDC1 interacts with ATM through its FHA domain causing a further accumulation of ATM at sites surrounding DSBs. This subsequently leads to phosphorylation of adjacent H2AX and additional binding of MDC1 and ATM. Through this mechanism the DNA damage signal is propagated and expanded over megabase chromosomal regions flanking DSBs [28, 33]. It is believed that the spread of γ-H2AX signal over the DSB-flanking chromatin is responsible for the observed increase in the size of IRIF [34, 35]. However, interaction of ATM with MDC1 appears not sufficient for its accumulation in IRIF because ATM fails to accumulate into IRIF without NBS1 suggesting that optimal recruitment of ATM requires NBS1 [36, 37]. This involves a direct interaction between the MRN complex and MDC1 and the subsequent binding of ATM to the C-terminus of NBS1. The interaction of MRN with MDC1 involves binding of the N-terminal FHA domain of NBS1 to phosphorylated Ser-Asp-Thr (SDT) repeats of MDC1 and provides a mechanism by which MRN accumulates at sites of DNA damage [15, 38]. The phosphorylation of the SDT repeats of MDC1 is mediated by casein kinase 2 (CK2). Finally, MDC1 provide a platform on which other DDR proteins are recruited. In addition to the accumulation of ATM and the MRN complex, MDC1 is essential for the accumulation of other DDR proteins to IRIF, including BRCA1 and 53BP1 [30, 31, 39-41]. MDC1 itself is phoshorylated by ATM at multiple adjacent TQXF motifs in its N-terminus [42]. These sites are critical for the recruitment of these DDR proteins to DSBs.

3. RAP80 and RNF8 are critical for cellular responses to DSBs

DDR proteins accumulate into IRIF with different kinetics. For example, in contrast to the early phosphorylation of H2AX and recruitment of MDC1, the translocation of BRCA1 and 53BP1 to the DNA damage foci occurs much later [40, 41] in agreement with the hierarchical process by which different DDR proteins are recruited and assembled to IRIF. Although MDC1 is indispensable for their translocation, the precise mechanisms by which many of the proteins are recruited remained still to be determined. Recent studies of RAP80, CCDC98/Abraxas, and RNF8 uncovered an intricate cascade of events in which several post-translational modifications, including phosphorylation and ubiquitination, co-operate in recruiting BRCA1 to IRIF.

3.1 RAP80 associates with BRCA1 and is required for the re-localization of BRCA1 to IRIF

BRCA1 is considered to play a key role in DDR and is of critical importance for the maintenance of genomic integrity by modulating checkpoints and DNA repair [43-47]. BRCA1 contains two BRCT motifs at its C-terminus and a RING domain at its N-terminus. This RING domain interacts with the RING-motif of BARD1 (BRCA1-associated ring domain protein 1) forming a heterodimer that functions as an E3 ubiquitin ligase. BARD1-BRCA1 heterodimers form several distinct complexes by interacting with different adaptor proteins, including BACH1 (BRCA1-associated C-terminal helicase), CtIP (CtBP-interacting protein), and CCDC98. These complexes are associated with distinct functions in DNA repair. BRCA1 translocates to IRIF several hours after DNA damage. This translocation is dependent on its BRCT domain; however, BACH1 and CtIP were found not to be responsible for recruiting BRCA1 to IRIF. On the contrary, their re-localization to IRIF was shown to depend on BRCA1 [48-50]. Recent studies have provided great insights into the mechanism by which BRCA1 is recruited to IRIF and have implicated several proteins in this process, including RAP80.

RAP80 (receptor-associated protein 80) or UIMC1 (ubiquitin-interaction motif containing 1) was identified as a novel partner of the BRCA1-BARD1 complex [51-54]. The RAP80 gene was initially cloned by our laboratory and shown to encode an acidic nuclear protein of 719 amino acids that is ubiquitously expressed and found most abundantly in testis and ovary. RAP80 was demonstrated to interact with the retinoid-related testis-associated receptor (RTR), also referred to as germ cell nuclear factor (GCNF) or NR6A1, a member of the steroid hormone nuclear receptor superfamily [55]. A recent study reported that RAP80 also interacts with the estrogen receptor α (ERα) in an agonist-dependent manner and is able to regulate ERα transcriptional activity [56]. In addition, RAP80 was found to interact with the SUMO-conjugating enzyme UBC9 (UBE2I) and shown to be a novel target for sumoylation [57]. The physiological significance of the interaction of RAP80 with UBC9 and whether this interaction has any relevance to DDR signaling needs further investigation.

Recent studies have demonstrated that RAP80 is associated with a BRCA1-BARD1-BRCC36 complex, which is abundant during M phase, and not with a BRCA1-BARD1-BACH1 complex that primarily occurs in S phase [53]. After IR and bleomycin treatment, RAP80 forms DNA damage foci that co-localize with those of BRCA1 [51-54]. Similar to BRCA1, RAP80 translocates to the DNA damage foci after a delay of 90-120 min [51], suggesting that RAP80 does not function as a DNA damage sensor but as a downstream mediator of DNA damage response signals. Formation of RAP80 foci depends on the presence of MDC1 and γ-H2AX [53, 54], but does not require a functional BRCA1 [51, 53, 54] in contrast to several other BRCA1-interacting proteins [49, 50]. Interestingly, knockdown of RAP80 expression greatly impairs formation of BRCA1 and BRCC36 IRIF, but not those of BACH1 [52-54]. These observations suggested that RAP80 might be a candidate for targeting BRCA1-BARD1-BRCC36 complexes to DSB sites.

3.2 RAP80 translocates to IRIF in a UIM dependent manner

In addition to two putative zinc finger motifs, RAP80 contains two UIMs at its amino terminus that exhibit high homology to the consensus UIM eeexΦxxAxxxSxxexxxx (in which Φ is a hydrophobic residue, e is a negatively charged residue, and x is any amino acid) [58, 59]. UIMs can mediate the monoubiquitination of proteins that contain these sequences, thereby regulating their activity and function. In addition, UIMs can recognize monoubiquitin and/or (poly)ubiquitin chains and, as such, mediate intra- or intermolecular protein interactions [58-61]. UIMs were first identified in the S5a subunit of the 19S proteasome complex, which recognizes ubiquitin chains of K48-linked polyubiquitinated proteins, thereby promoting their entry into the proteasome barrel and their subsequent degradation [62]. The UIMs of RAP80 were shown to bind (poly)ubiquitin directly and to stimulate multi-ubiquitination of RAP80 [53, 54, 56], showing that these UIMs are functional. The RAP80 UIMs exhibit a preference for K6- or K63-linked ubiquitin chains and have low affinity for K48-linked ubiquitin chains [53, 54]. Interestingly, these functional UIMs are required for the re-localization of RAP80 to IRIF after IR treatment, because a RAP80 mutant lacking the UIMs failed to translocate to the DNA damage foci [51-54]. Moreover, mutation of critical amino acids in the UIMs greatly disrupted the formation of RAP80 IRIF [51, 54]. These observations led to the hypothesis that the translocation of RAP80 and associated proteins is mediated through an interaction of UIMs with one or more K6- or K63-linked (poly)ubiquitinated proteins at or near DSB sites. This concept was supported by findings of Sobhian et al. demonstrating that HA-tagged polyubiquitin co-localizes with BRCA1 IRIF after DNA damage and that only K6- or K63-linked ubiquitin chains, but not K48-linked ubiquitin chains, form IRIF [53]. Similar to BRCA1 and RAP80, the formation of ubiquitin IRIF depended on MDC1 and γ-H2AX. These observations indicated that DNA damage induces ubiquitination of one or more proteins at the sites of DNA damage consistent with the hypothesis that UIMs of RAP80 target BRCA1 complexes by recognizing K6-or K63-linked ubiquitin chains at DSB sites [53, 54].

3.3 RAP80 is important for both G2/M phase checkpoint and optimal HRR repair

Because RAP80 associates with BRCA1 and recruits BRCA1 to the DNA damage sites, one might predict a potential role for RAP80 in the regulation of certain damage responses. The latter was supported by studies showing that RAP80 depletion increased the sensitivity of cells to IR-induced cell death [51-54]. Moreover, RAP80-depleted cells showed impaired IR-induced CHK1 activation [54], and as a consequence, defective G2/M phase checkpoint control [52-54]. Moreover, RAP80 was found to be required for optimal HRR of DSBs [51, 52]. These observations indicate that RAP80 has a regulatory function in DNA damage response signaling, probably by facilitating the recruitment of BRCA1-BARD1-BRCC36 complexes to DNA damage foci. However, the effects of RAP80 knockdown were not as dramatic as those observed by depletion of BRCA1 [52], suggesting that the interaction of RAP80 with ubiquitinated proteins at the sites of DNA damage may not be the only mechanism to recruit BRCA1. Whether RAP80 is also involved in repair of DSBs through NHEJ needs to be determined.

3.4 CCDC98/Abraxas bridges RAP80-BRCA1 association

About 50% of familial breast cancer and 20-30% of hereditary ovarian cancer are associated with mutations in the BRCA1 gene [63]. Many of these mutations are within the two BRCA1 C-terminal (BRCT) repeats of BRCA1 [64]. BRCT domains have been identified in several proteins and shown to mediate protein-protein interactions by binding pSer(Thr)-X-X-Phe motifs [65, 66]. These BRCT repeats are essential for the tumor repressor functions of BRCA1 and required for its translocation to IRIF. Moreover, the BRCA1-BRCT repeats are necessary and sufficient for its association with RAP80 [51-54]. Mutations within the BRCT repeats, found to be associated with breast/ovarian cancer, disrupted the association of BRCA1 with RAP80 [53, 67]. These observations support the conclusion that the association of RAP80 with BRCA1 involves the BRCT repeats; however, RAP80 does not contain any functional pSer(Thr)-X-X-Phe motifs that could bind BRCT [67], suggesting that the interaction of RAP80 and BRCA1 is indirect and mediated through another BRCT-associated protein.

Recently, a new protein called coiled-coil domain-containing protein (CCDC98) or Abraxas was identified as a new BRCA1-BRCT interacting protein [52, 68, 69]. BRCA1-BRCT interacts directly with CCDC98/Abraxas by binding the Ser-Pro-Thr-Phe phosphopeptide motif at its C-terminus. Point mutations in this motif greatly diminished the interaction of CCDC98/Abraxas with BRCA1-BRCT; however, this interaction occurred independently of DNA damage [52, 68, 69]. Both CCDC98 and BRCA1 translocate and co-localize to IRIF after DNA damage. This translocation involves a direct interaction between CCDC98/Abraxas and RAP80 that is phosphorylation-independent and mediated by the N-terminal region of CCDC98/Abraxas and an internal region of RAP80 (aa 235-337) [52, 69]. Interestingly, knockdown of CCDC98/Abraxas expression disrupted the translocation of BRCA1 to IRIF suggesting that its translocation was dependent on its interaction with CCDC98/Abraxas. In contrast, knockdown of CCDC98/Abraxas did not effect the re-localization of RAP80 to DNA damage sites, whereas the formation of both BRCA1 and CCDC98/Abraxas foci were abolished in RAP80-depleted cells [68, 69]. These observations suggested that RAP80 is associated with the BRCA1 protein complex through its interaction with CCDC98/Abraxas and that it facilitates the recruitment of both CCDC98/Abraxas and BRCA1 to IRIF by recognizing K63-linked ubiquitin structures at DNA damage sites. Cells, in which CCDC98/Abraxas expression was knocked down by siRNAs, showed increased sensitivity to IR, reduced CHK1 phosphorylation, and impaired G2/M checkpoint activation after DNA damage, suggesting that, as RAP80, CCDC98/Abraxas is an upstream regulator of BRCA1 function [52, 68, 69].

3.5 RAP80 recognizes histones ubiquitinated by RNF8

Next, research efforts focused on identifying the missing links, particularly the identification of the E3 ubiquitin ligase, E2 ubiquitin-conjugating enzymes, and their target proteins, involved in mediating the interaction between the UIMs of RAP80 and K63-linked polyubiquitin chains. These studies led to the identification of RNF8 as a DNA damage-responsive protein that plays a key role in facilitating this interaction [42, 70-72]. RNF8, a ubiquitin ligase with an FHA domain at its N-terminus and a RING-finger domain at its C-terminus, was found to bind class III ubiquitin conjugating enzymes through its RING-finger domain [73]. RNF8 was initially reported to interact with the retinoid X receptor α (RXRα) through their N-terminal domains and to promote its transcriptional activity [74]. Through a siRNA library screen, Kolas et al. found that RNF8 depletion greatly inhibited the formation of 53BP1 IRIF [42], suggesting that RNF8 is involved in DDR signaling and required for 53BP1 translocation. Moreover, RNF8 foci are formed rapidly after DNA damage, while other E3 ligases, including Chfr and Znrf2, do not translocate to IRIF [71, 75]. Formation of RNF8 IRIF depends on the presence of H2AX and MDC1, while NBS1, BRCA1, 53BP1, and RAP80 are not required [71], indicating that RNF8 functions at an early stage in DDR and downstream of H2AX and MDC1. Subsequent analysis demonstrated that RNF8 directly interacts with MDC1 and that its FHA domain recognizes a cluster of TQXF ATM phosphorylation target sites in the N-terminus of MDC1 [70-72, 76]. Mutation of all these ATM motifs is required to totally abolish the interaction with RNF8.

Further studies showed that RNF8 depletion caused irradiation hypersensitivity, impaired G2/M checkpoint control, and a disruption of RAP80, BRCA1, and 53BP1 foci formation [42, 70-72]. Intriguingly, reconstitution with a mutant RNF8, in which the RING-finger domain was deleted, did not restore RAP80 and BRCA1 foci formation, although this RNF8 mutant itself was able to go to the foci [70, 71]. These observations indicated that both the FHA domain and the RING-finger domain, which contains the ligase activity, of RNF8 are critical for the recruitment of RAP80 and BRCA1 to IRIF. In addition, RNF8 depletion markedly impaired IRIF formation of conjugated ubiquitin [42, 70-72]. These findings supported the concept that RNF8 mediates the K63-linked ubiquitination of specific proteins thereby facilitating their interaction with the UIMs of RAP80. However, RNF8 must act together with an E2 ubiquitin-conjugating enzyme to ubiquitinate target proteins. UBC13 was identified as a good candidate because it catalyzes K63-linked polyubiquitination [77]. Subsequent studies showed that although RNF8 is able to interact with multiple ubiquitin-conjugating enzymes (E2s) to stimulate K48- and K63-linked ubiquitin conjugation, UBC13 was the only enzyme capable of catalyzing K63-linked ubiquitination [73, 78]. Depletion of either RNF8 or UBC13 impaired IRIF formation of conjugated ubiquitin and disrupted recruitment of BRCA1 and RAP80 to DNA damage foci [42, 71, 72]. These observations suggested that at DNA damage sites UBC13-RNF8 complexes catalyze the formation of K63-linked ubiquitin chains that are subsequently recognized by RAP80. The importance of UBC13 in DDR was further indicated by recent observations showing that depletion of UBC13 increased the sensitivity of cells to irradiation and compromised DSB repair by HRR [79].

The next question was: which proteins function as targets of UBC13? Several studies identified H2A and H2AX as RNF8 substrates at the sites of DNA damage and further established the link between their ubiquitination and RAP80 [70, 71]. These studies demonstrated that RNF8 physically interacted with H2A and stimulated its ubiquitination. This ubiquitination was enhanced by IR. MDC1 knockdown compromised IR-induced H2A ubiquitination in agreement with the model that RNF8 needs to be recruited by MDC1 at the damage foci in order to ubiquitinate H2A [70]. H2AX is ubiquitinated by RNF8 to a similar extent as H2A and requires H2AX phosphorylation by ATM, because Ser139 mutated H2AX is much less ubiquitinated after IR [71]. These results support a model in which RNF8 ubiquitinates H2A and H2AX that then act as binding sites to recruit RAP80 and BRCA1. However, H2A and H2AX are also ubiquitinated under normal conditions [70, 71]; however, these ubiquitin conjugates may not be recognized by RAP80. Alternatively, these sites may not be accessible for RAP80 before IR but become accessible after IR when RNF-8-mediated histone ubiquitination induces alterations in chromatin configuration and expose the pre-existing ubiquitin structures to RAP80 [70]. This could then further expand BRCA1 recruitment to regions flanking DSBs.

3.6 RNF8 is critical for 53BP1 retention at DSB sites

53BP1 was first reported to interact with the tumor suppressor p53 and to stimulate its activity [80, 81]. In addition, 53BP1 functions as key transducer in DNA damage signaling and is required for both IR-induced G2/M phase and intra-S-phase checkpoints [82]. It contains two BRCT motifs that mediate the interaction with p53 and two tandem Tudor domains, which bind methylated histones H3 or H4 and target 53BP1 to DSBs [83-86]. MDC1 and RNF8 and its ligase activity are critical in the recruitment of 53BP1 to IRIF, [42, 70, 71], suggesting that ubiquitination of H2A and H2AX by RNF8 is required for the translocation of 53BP1 to the sites of DNA damage. However, the translocation of 53BP1 is independent of RAP80 [54]. It has been proposed that interaction of γ-H2AX-MDC1-RNF8 and the subsequent ubiquitination of histones H2A and H2AX may promote additional modifications in proteins, which together induce changes in chromatin conformation at DSB-flanking regions and possibly increased accessibility of methylated DNA to 53BP1 [70].

3.7 RAP80 recruits deubiquitinase BRCC36 to DSBs

Analysis of BRCA1-BARD1 protein complexes by mass spectrometry identified BRCA-containing complex (BRCC) subunits BRCC36 and BRCC45, as BRCA1-BARD1 interacting proteins [50, 87]. BRCC36 displays sequence homology to CsnE/Jab1, the fifth subunit of COP9 signalosome, including a conserved JAMM motif. These motifs have been found to be associated with proteins that function as deubiquitinases [87, 88]. Depletion of BRCC36 and BRCC45 in cells increased their sensitivity to IR, compromised IR-induced G2/M checkpoint control, and prevented BRCA1 phosphorylation and translocation to IRIF after IR [87, 89]. Subsequent studies showed that BRCC36 is part of a BRCA1-BARD1 protein complex with CCDC98/Abraxas and RAP80 [53]. BRCC36 interacts directly with CCDC98/Abraxas through their coiled-coil domains while RAP80 associates with BRCC36 by directly binding CCDC98/Abraxas [53, 72]. RAP80 is essential for the translocation of BRCC36 and CCDC98/Abraxas while depletion of BRCC36 significantly decreases foci formation of CCDC98/Abraxas [72]. Possibly, BRCC36 is needed for optimal interaction between CCDC98/Abraxas and RAP80.

Recently, BRCC36 was found to have a deubiquitinase (DUB) activity on K63-linked ubiquitin chains [53], which are recognized by RAP80 and mediate the recruitment of RAP80 and BRCA1 to the damage foci. What role the DUB activity of BRCC36 plays in the DNA repair process needs further investigation. It has been suggested that BRCC36 might be involved in the deubiquitination of certain chromatin-associated proteins, leading to changes in chromatin structure and amplification of damage signals. BRCC36 may also play a role in the deubiquitination of proteins ubiquitinated by BRCA1/BARD1 and RNF8 ligases in order to turn off DNA damage signals after the damage is repaired, which is required to resume normal DNA replication, cell proliferation, and gene transcription.

3.8 RAP80 is an ATM target protein

Many proteins involved in IR-induced DNA damage responses are targets of phosphorylation by ATM [90]. The sequence S/TQ has been identified as the minimal consensus phosphorylation site of all PI3K-like family members [91]. Human RAP80 contains 8 potential ATM phosphorylation sites [51], 5 of which (Ser101, Ser140, Ser205, Ser402, and Ser419) were reported to be phosphorylated by ATM in vitro or in living cells [51, 53, 54, 67, 92]. None of these phosphorylation sites of RAP80 are highly conserved among different species [52]. Interestingly, RAP80 is also phosphorylated by ATR after UV treatment and re-localizes to UV-induced damage sites [67], suggesting a more general role of RAP80 in checkpoint control and repair of different forms of DNA lesions.

RAP80 is phosphorylated by ATM within 5 min after IR, much earlier than its accumulation at DNA damage foci [67]. Although RAP80 is part of a BRCA1 protein complex and recruits this complex to IRIF, BRCA1 itself is phosphorylated by ATM after it translocates to the damage foci [36]. Whether this early phosphorylation of RAP80 by ATM has a specific regulatory function has yet to be determined. Phosphorylation can affect protein-protein interaction and the activity/function of proteins. Mutation of multiple ATM phosphorylation sites in RAP80 did not affect the translocation of RAP80 or BRCA1 to IRIF. Therefore, unlike activating transcription factor 2 (ATF2), which is also rapidly phosphorylated by ATM after DNA damage [34], the translocation of RAP80 to IRIF is independent of ATM phosphorylation [51, 53]. Thus, phosphorylated RAP80 might have additional functions in DDR besides targeting BRCA1 to the DNA damage foci.

4. Conclusions and questions to be answered

Studies discussed in this overview have led to a hierarchical model (Figure 1) in which phosphorylation and ubiquitination regulate a series of protein-protein interactions that provide a mechanism by which several protein complexes are assembled and recruited to DNA damage sites [42, 51-54, 56, 67-72]. The rapid activation of ATM and phosphorylation of H2AX is one of the earliest events after the induction of DSBs. This leads to the binding of MDC1, which recognizes a phosphorylated peptide in the C-terminus of γ-H2AX through its BRCT domain. MDC1 appears to have multiple functions. It protects γ-H2AX from dephosphorylation and facilitates the recruitment of additional MRN and ATM proteins that subsequently leads to the phosphorylation of additional H2AX and MDC1. This involves a direct interaction between the MRN complex and MDC1 and the subsequent binding of ATM to the C-terminus of NBS1. In this manner, the DNA damage signal is propagated and expanded over megabase chromosomal regions flanking DSBs. Phosphorylated MDC1 is also recognized by RNF8 through its FHA domain that then with the aid of UBC13 catalyzes the K63-linked ubiquitination of H2A and H2AX.

Fig. 1Fig. 1
Schematic illustration of the hierarchical functions of ATM, H2AX, MDC1, RNF8 UBC13, CCDC98/Abraxas, and RAP80 in the recruitment of BRCA1-BARD1 protein complexes to DSB sites. (A) Induction of DSBs by ionizing irradiation (IR) results in the recruitment ...

The UIM containing protein RAP80 plays a key role in the recruitment of BRCA1 protein complexes to DSBs [51-54]. RAP80 is part of the BRCA1-BARD1 protein complex by binding CCDC98/Abraxas, which itself is bound directly to BRCA1-BARD1. The UIMs of RAP80 bind K63-linked ubiquitin chains and therefore are able to recognize ubiquitinated H2A/H2AX, resulting in the recruitment of the entire complex to DNA damage foci. However, other proteins are recruited to IRIF by different mechanisms. For example, the translocation of 53BP1 to methylated histones at DNA damage foci is dependent on the ubiquitination of H2A/H2AX by RNF8, but is independent of RAP80 [42, 70, 71]. The role of this ubiquitination in 53BP1 recruitment is not quite understood but may involve RNF8-mediated ubiquitination of histones and subsequent remodeling of the chromatin structure that makes methylated histones more accessible to 53BP1.

Although these studies greatly advanced our understanding of the mechanisms by which several DDR proteins are recruited and accumulate at DSB sites, many questions remain unanswered. After induction of DSBs, RAP80 is rapidly phosphorylated by ATM; however, this phosphorylation does not have any apparent role in the translocation of either RAP80 or BRCA1 [51-54, 67]. Therefore, this phosphorylation might regulate a different function of RAP80 and possibly modulate the interaction of RAP80 with other proteins. In addition to CCDC98/Abraxas, RAP80 interacts with the estrogen receptor ERα in an agonist-dependent manner and modulates the transcriptional activity of ERα [56]. ATM phosphorylation might affect the interaction of RAP80 with ERα. Interestingly, ERα has also been found to interact with BRCA1 and to function as a substrate for BRCA1 ubiquitin ligase [93-95]. Whether there is any relationship between the interaction of RAP80 with BRCA1 and the link between these proteins and ERα signaling pathway needs further investigation.

Protein complexes formed at DNA damage sites have to be removed after the damage is repaired. Although little is understood about this process, it likely involves dephosphorylation of α-H2AX and other proteins, K63-linked deubiquitination, and ubiquitination-proteasome dependent degradation. It is therefore interesting that BRCA1/BARD1, which functions as an E3 ligase, is associated with the deubiquitinase BRCC36 [53, 96]. It has been proposed that the deubiquitinase activity of BRCC36 might play a role in terminating the DNA repair signaling later in DDR. However, whether RAP80 and CCDC98/Abraxas play a role in the regulation of BRCA1 ligase and/or BRCC36 deubiquitinase activity is not well understood.

It is well established that BRCA1 functions as a tumor suppressor [1, 13]. Mutations in the BRCA1 gene account for about 50% of familial breast cancers and 20%-30% of hereditary ovarian cancers [63]. Based on the roles that RNF8, RAP80, and CCDC98/Abraxas have in DDR, one might expect that mutations in genes that would compromise BRCA1 activity, are implicated in breast and ovarian cancer. A recent study found that 5-10% of patients with different types of cancer contain specific antibodies against RAP80 and it was concluded that RAP80 might function as a new cancer-associated antigen [97]. However, mutational analysis of RAP80 and CCDC98/Abraxas genes in 168 multiple-case breast/ovarian cancer families, negative for mutations in BRCA1 or BRCA2, showed only few (10 and 6, respectively) variants most of which are considered common [98]. A different study analyzing RAP80 mutations in 152 women with familial breast cancer, negative for BRCA1 and BRCA2 mutations, identified several missense but no truncating mutations [99]. These studies suggest that RAP80 and CCDC98 do not play an important role as high penetrance breast cancer susceptibility genes; however additional studies are needed to determine whether these proteins have any role in human cancer.

Within the last year great advances have been made in understanding the function of RAP80 and RNF8 in facilitating the recruitment of the BRCA1-BARD1-BRCC36-CCDC98 protein complex to IRIF. Future studies have to determine whether these proteins play a role in other cellular processes in which DDR is critical, including cellular senescence [100] and telomere maintenance [101].

Acknowledgements

We would like to thank Drs. Sonnet Arlander and Daniel Menendez for their valuable comments on the manuscript. This research is supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

Footnotes

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