The p53 gene encodes a tumor suppressor whose primary function is in transcription. p53 is inactivated or disrupted in ≥50% of all human cancers. Mdm2, an E3 ubiquitin ligase, interacts with the N-terminus of p53 and ubiquitinates it, thus marking the protein for destruction by the proteosome. ATM phosphorylates p53 in response to DSBs, an event that prevents its Mdm2-mediated degradation and results in the stabilization and accumulation of the protein 12. Using the core DNA binding domain of p53 (residues 80–320) as bait in a two hybrid screen, Fields and colleagues first identified 53BP1 in 1994 3. Human 53BP1 is comprised of 1,972 residues and contains important structural elements including two Breast Cancer Gene 1 (BRCA1) C-terminal (BRCT) repeats, tandem Tudor domains, a GAR methylation stretch, two dynein light chain (LC8) binding sites, and numerous PIK kinases and cyclin-dependent (CDK) phosphorylation sites (Fig. 1). The sequences of 53BP1 that bind p53 include the C-terminal BRCT region. In vitro, the tandem BRCT repeats of 53BP1 (residues 1,724–1,972) bind core p53 residues with a K_d of 6 μM as determined by isothermal titration calorimetry 4. First identified in BRCA1, BRCT motifs have been identified in a number of proteins that are connected to DNA damage response mechanisms. BRCT motifs have been reported to participate in various processes such as transcriptional activation and they have the capacity to serve as phospho-peptide binding modules 56. Because wild-type, but not mutant p53 (i.e. R175H) binds 53BP1, the conformation of p53 appears crucial for the 53BP1-p53 interaction 3. To date, p53 is the only factor reported to directly interact with any of the two BRCT motifs of 53BP1. Subsequent transient co-transfection experiments with 53BP1 and p53 reporter plasmids suggested that 53BP1 enhanced p53-mediated transcription 7. Another report suggesting a link between 53BP1 and transcription came with the identification of a 98 amino acid region of murine 53BP1 (corresponding to human residues 1,179–1,277) that interacted with the p202 transcription factor 8. The significance of this interaction remains uncertain.

The crystal structure of the recombinant BRCT motifs of 53BP1 and the central DNA binding domain of p53 (core) has been solved 910. Here, p53 binds to the N-terminal BRCT motif and the linker region of 53BP1. Importantly, the structural analysis also reveals that the same p53 residues are involved in binding both 53BP1 and DNA, making it very difficult to imagine how 53BP1 could enhance p53-mediated transcription. This point has been previously discussed by Halazonetis and co-workers 11. Although it appears very unlikely that 53BP1 enhances p53-mediated transcription as once suggested, one report has concluded that 53BP1 positively regulates the BRCA1 promoter 12. In this study, the p53-proficient U20S cell line was co-transfected with siRNA molecules directed against 53BP1 and a luciferase reporter construct under the control of the minimal BRCA1 promoter. This resulted in >70% inhibition of promoter activity 12. Furthermore, using chromatin immunoprecipitation (ChIP) assays, 53BP1 was shown to bind to an imperfect palindromic sequence within the BRCA1 promoter element. How 53BP1 regulates BRCA1 expression remains unknown, but it is possible that its BRCT repeats possess transactivation potential.

In response to IR, the histone variant H2AX is rapidly phosphorylated by PIK kinases and relocalizes to sites of DNA damage 2223. The accumulation of phosphorylated histone H2AX (γ-H2AX) at and near sites of DSBs can be detected cytologically as irradiation induced foci (IRIF) 2223. Using antibodies against human and Xenopus 53BP1, several labs found that 53BP1 forms IRIF in response to genotoxic stress, particularly agents that induce DSBs 24252627. Such foci overlap with those previously described for H2AX and Mre11. Moreover, 53BP1 possesses numerous PIK kinase phosphorylation sites (S/TQ) and was shown to be a substrate for ATM and probably its related kinases 242527. 53BP1 phosphorylation, however, is not required for IRIF formation 28, although it clearly represents an activating event. Based upon these observations, 53BP1 was proposed to operate in DSB repair. Indeed, 53BP1 has been shown to physically associate with a number of proteins involved in various aspects of DSB repair (Table 1). A further glance into the functional role of 53BP1 in DNA damage signaling came with the analysis of mammalian cell lines depleted in 53BP1 expression through siRNA or from knock-out mice (see below) 132930. These cells revealed that 53BP1 was required for the optimal, DNA damage-inducible phosphorylation of ATM substrates such as Smc1, Chk2, and BRCA1 132930. Furthermore, 53BP1-deficient cells have been reported to have a mild intra-S phase (RDS phenotype) and a nearly three-fold defect in the G₂/M checkpoint, but only at low doses of IR 1329. One study showed that 53BP1^-/- cells had a prolonged G₂ phase, a phenotype also reported in ATM-deficient cells 30. This suggested that 53BP1 mediated the DNA damage response by facilitating ATM activity towards at least a subset of its substrates and raised the possibility that 53BP1 could function in ATM activation, an event that is likely to be tightly coupled to chromatin structure and function 1129.

How cells sense and respond to DSBs has been the subject of intense research by numerous laboratories. Because the DNA repair apparatus uses damaged DNA as a substrate, the DNA damage response must be tightly linked to chromatin structure and function. In response to DSBs, the Mre11/Rad50/Nbs1 (MRN) complex is rapidly recruited to sites of DNA breaks 3132. In budding yeast, the Mre11 complex facilitates nucleosomal displacement near the DSB and H2A phosphorylation 33. Here, loss of H2B and H3 also depends on the Ino80 chromatin remodeling complex 33. In fact, ChIP analysis reveals that phosphorylated H2A in budding yeast is not associated with the break per se, but rather the Mre11 complex appears to localize to sites of DNA damage 34. This is believed to contribute, at least in part, to ATM activation 3536. In addition, autophosphorylation of ATM, particularly at S1981 (S1981-P), has been linked to its activation where it occurs in response to DNA damage and changes in chromatin structure 37. However, the role of S1981 phosphorylation is not clear because mice deficient in the mutant appear largely normal for several ATM functions, including those that occur in B and T cells 38. ATM activation, as measured by S1981 autophosphorylation, also occurs in response to changes in chromatin structure in the apparent absence of DNA breaks 37. Moreover, the MRN complex is also believed to contribute to the activation of ATM kinase 3536. Acetylation of ATM by Tip60 has also been attributed to its activation 39. Thus, ATM activation appears to be a highly regulated event where autophosphorylation at S1981 may represent just one facet of its activation.

ATM phosphorylates and contributes to the activation of a number of important DNA damage response regulators. In response to IR, ATM phosphorylates 53BP1 on serine residues 25 and 29 28. Importantly, ATM phosphorylates the C-terminal tail of H2AX, generating γ-H2AX which directly recruits and binds Mdc1 (formerly known as NFBD1), a factor that, like 53BP1, contains two C-terminal BRCT repeats in addition to a forkhead domain 404142. Through the BRCT motifs of Mdc1, the γ-H2AX-Mdc1 complex recruits another ATM molecule, thus amplifying the original DNA damage response signal through the creation of a positive feedback loop 4041424344. As a consequence, neighboring H2AX molecules that reside in a subset of histone octamers peripheral to the DNA break become phosphorylated, resulting in the generation of γ-H2AX molecules that lie up to one megabase from the actual site of the DNA break.

A variety of proteins, including 53BP1, have been shown to localize to IRIF in many cell types. In lymphoid B cells, 53BP1 localizes to the switch locus of murine chromosome 12 1445. The precise biological role of IRIF is unknown, but they are in all probability associated with DNA repair. Indeed, the accumulation of H2AX to IRIF is routinely used as a marker for DSBs. Because many cancer cells and tissues exhibit constitutive 53BP1 foci, activation of DNA damage response pathways may be an early feature in human tumorigenesis, probably as a consequence of the intrinsic genomic instability in these cells 4647. This could provide a selection for p53 loss during tumorigenesis and has been proposed to explain the large frequency of p53 mutagenesis in cancer 47. Indeed, loss of 53BP1 function correlates with cancer progression in human tumors 4647. How widespread 53BP1 dysfunction is in human tumors has not been thoroughly addressed. Interestingly, a human patient with a myeloproliferative disorder was found to have a translocation that generated a fusion protein between 53BP1 and the platelet-derived growth factor (PDGF; 48). Because, in this scenario, PDGF tyrosine kinase activity depends on the ability of its fusion partner sequences to form oligomers, it was proposed that 53BP1 could form oligomers 48. Indeed, 53BP1 oligomerizes in vivo, but the significance of this remains unknown 49.

Although some siRNA studies concluded that 53BP1 and Mdc1 IRIF formation occurs independently of each other 405051, it is now clear that both γ-H2AX and Mdc1 are required for the accumulation of 53BP1 to IRIF 414252. In addition, the histone acetyltransferase (HAT) co-factor Trrap and HAT Tip60 complex (Trrap-Tip60) is required for 53BP1 IRIF formation 53. Trrap-Tip60 binds to chromatin near DSBs and Trrap-deficient cells are impaired in break-induced HR 53, underscoring the intimate link between DNA repair and chromatin structure (see below). Since Trrap is required for 53BP1 IRIF formation and influences HR 53, does this necessarily mean that 53BP1 functions in break-induced HR? Apparently not, as 53BP1 plays no role in this process when tested with in an I-Sce I-based system developed in the Jasin laboratory that measures gene conversion 54. Despite this, ChIP experiments indicate that 53BP1 localizes with the Rad51 paralogs Rad51A and Rad51C to I-SceI-induced DSB foci 55. Moreover, in the absence of extrinsic DNA damage, 53BP1 localizes to "nuclear dots" during S/G₂ 53, a phenomenon similarily reported for Rad51 and BRCA1 56. Unlike 53BP1, H2AX and Mdc1 have been reported to participate in HR 545758, further illustrating the differences between regulators of DSB repair pathways. 53BP1^-/- fibroblasts, however, display a hyper-rec phenotype, suggesting that the protein suppresses aberrant, spontaneous recombination (as opposed to break-induced HR). Clearly, 53BP1 does not act redundantly with H2AX and Mdc1 during the DNA damage response.

Although unlikely to be a structural component of chromatin, 53BP1 possesses several intriguing links to the biology of chromatin, particularly with respect to the regulation of histone modifications 5960. IR-inducible phosphorylation of 53BP1, which likely represents the "active" protein, associates with chromatin 61. In terms of CSR in B cells, one possibility is that 53BP1 synaptic function provides a scaffold for coordinating DNA repair through modulating chromatin structure in the language of a histone code. First, in addition to the well documented role of γ-H2AX-dependent recruitment of 53BP1 to IRIF, 53BP1 possesses tandem Tudor motifs (53BP1^TT) 5960 which have been reported to associate with various methylated lysine residues in histones H3 and H4 6062. This includes lysines K4, K9, and K20 in histone H4 and H3 K79 (by the Dot1 methyltransferase). Intriguingly, in fission yeast, methylation of histone H4 at K20 has been linked to DNA repair through its interaction with Crb2 63, a potential 53BP1 orthologue (see below). No such function for H4 K20 has been reported in mammalian cells. Tudor motifs resemble chromodomains at the structural level and associate with methylated arginine and lysine residues 64. For example, 53BP1^TT, the minimal region of the protein sufficient for IRIF formation, binds to H3 K79-CH₃ independently of DNA damage 60. Although this modification is constitutive, lysine 79 is sterically inaccessible under normal conditions as it lies in the nucleosome core. Because of this, 53BP1 has been proposed to sense changes in chromatin structure that occur "upstream" of the DNA break 60. In this regard, a perturbation in chromatin structure, as introduced through a nearby DSB, is thought to expose H3-K79-CH₃ and allow for 53BP1 binding and execution of its essential functions in DSB repair. Indeed, 53BP1 apparently binds H3-K79-CH₃ under conditions that perturbed chromatin structure without introducing DSBs 60. In the case of isotype switching in activated B cells, 53BP1 activation could be induced through transcriptional events that alter chromatin structure prior to the genesis of AID-dependent DNA breaks. Thus, recruitment of 53BP1 to chromatin during DSB repair may proceed through multiple histone modifications and is likely connected to its role in a non-classical form of end joining ("anchoring") 65.

As it operates early in the DNA damage response and associates with modified histones, 53BP1 is a strong candidate for participating in both DSB repair and chromatin remodeling in the vicinity of the break or in response to other types of chromatin perturbation. In this manner, DNA repair by 53BP1 is coordinated with IRIF factors like H2AX and Mdc1 in conjunction with its ability to influence chromatin architecture. Previous studies have indicated that in response to DNA damage yeast H2A recruits chromatin remodeling complexes 666768. Additional, non-mutually exclusive mechanisms may also be operative in this process. For example, the PRMT1-dependent methylation of 53BP1 at arginine residues 4969 within its GAR motif (Fig. 1) may form an epigenetic signal that participates in 53BP1 function. However, GAR methylation does not influence DNA binding or IRIF formation by 53BP1 49. Furthermore, 53BP1 protein levels are reciprocally regulated by the histone deacetylase 4 (HDAC4; 68). Because one might anticipate that chromatin near a DSB would exist in a "relaxed" form that permits the DNA repair apparatus access to the sites of damage 70, perhaps 53BP1 activity influences acetylation levels through HDAC4 function.

On first glance, there appear to be no obvious orthologues of 53BP1 in yeast. However, Rad9 and Crb2 in budding and fission yeast, respectively, possess similar functional elements (Tudor and BRCT) and parallel properties with 53BP1, despite low overall sequence homologies. Therefore, DNA damage response mechanisms in yeast will be instructive for understanding mammalian 53BP1 function. For example, Rad9 is phosphorylated in response to DNA damage by yeast PIK kinases 97. Interestingly, loss of Dot1 (and therefore methylation of H3-K79) prevents activation of Rad9 and decreases its localization to sites of DSBs 98. Inhibition of IR-dependent phosphorylation of H2A yields cells with very minor checkpoint defects 6399100. Despite some interesting parallels between 53BP1 and Crb2, there are some distinct differences. For example, the BRCT motifs of Crb2 are required for both homo-oligomerization and IRIF formation 101, but the sequences sufficient for this in 53BP1 lie upstream of the GAR motif and in the Tudor domains, respectively (Fig. 1).

As in higher eukaryotes, phophorylation of yeast histone 2A (γ-H2A), occurs rapidly in response to DSBs, indicating that this event is highly conserved. The yeasts, however, do not possess orthologues to Mdc1 and BRCA1. Yeasts defective in H2A phosphorylation have, at best, mild defects in NHEJ and checkpoint signaling 6399. Rad9, Crb2, and 53BP1 (at least at low doses of IR) are required for cell cycle arrest in response to DNA damage 1328102. In fission yeast γ-H2A recruits Crb2 to sites of DNA damage 102. Interestingly, the accumulation of Crb2 at IRIF in fission yeast is dependent on both γ-H2A and histone H4 methylation at K20 63103, a modification that plays a role in DNA repair in fission yeast 63. Crb2 IRIF formation appears independent of Dot1 activity as this methyltransferase does not appear to exist in the fission yeast genome 103. Furthermore, in the absence of both H2A and H4 K20 histone modifications, Crb2 activity remains largely functional 103. Here an alternate recruitment pathway appears sufficient for IRIF formation and checkpoint activation. Such a pathway requires CDK phosphorylation of Crb2 at T215. Crb2 phosphorylation at T215 phosphorylation is important for its association with the BRCT protein Cut5, an orthologue of mammalian TopBP1 100. Moreover, hypomorphic crb2-T215A cells have checkpoint maintenance defects 100102. Therefore, multiple DNA damage signals are programmed into Crb2 function, allowing it to coordinate DNA repair with cell cycle progression and chromatin remodeling.

53BP1 plays an important role in DNA damage signaling pathways through its ability to influence the function of a variety of factors including ATM and p53 (Fig. 3). Importantly, 53BP1 participates in DNA repair through its role as a tumor suppressor protein, particularly in cells of B and T origin. Here, 53BP1 collaborates with H2AX and Mdc1 to participate in a novel form of end joining termed "anchoring". In this setting, 53BP1 is recruited to or near sites of DSBs where it coordinates DNA repair in the context of chromatin structure, most likely through the language of a histone code. Whether 53BP1 participates in chromatin remodeling events remains an open question. Given its large size, multiple domains and various interacting partners, 53BP1 is well poised to operate as a mediator in multiple DNA damage signaling events.