DNA repair pathway choice—a PTIP of the hat to 53BP1

In the June issue of Cell, Nussenzweig and colleagues identify PTIP/PAXIP as a 53BP1 effector protein in the regulatory network that controls DSB repair pathway choice.Cell (2013) 153 6, 1266–1280 doi: 10.1016/j.cell.2013.05.023DNA double-stranded breaks (DSBs) are highly cytotoxic lesions that can induce genome rearrangements if not accurately repaired. DSBs can be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is the preferred repair pathway during the S and G2 cell cycle phases because a sister chromatid provides a perfect template for ‘error-free'' repair. During G1, when HR is suppressed to prevent recombination with homologues, repair is achieved primarily by NHEJ. Molecularly, DSB repair pathway choice is largely regulated at the level of 5′ to 3′ DNA end resection, that is, the formation of the 3′ end single-stranded DNA overhangs that are used to initiate HR. End resection inhibits NHEJ and promotes HR.In the June issue of Cell, Nussenzweig and colleagues identified the protein PTIP (also known as PAXIP) as a new component of the regulatory network that controls DSB repair pathway choice [1]. This work has important implications for our understanding of the mechanisms by which genomic integrity is underpinned, and is especially germane to those interested in the genesis of breast and ovarian cancer caused by a defective BRCA1 protein, which is crucial for DSB repair by HR.53BP1 (also known as TP53BP1) is a key determinant of DSB repair pathway choice [2]. In response to DSBs, 53BP1 binds to chromatin at damaged sites, where it promotes NHEJ by blocking end resection. 53BP1 has a crucial role during class switch recombination (CSR) in B cells and the fusion of dysfunctional telomeres. An even more striking phenotype was observed in mice in which loss of 53BP1 reversed most of the phenotypes associated with BRCA1 deficiency, including cell and embryonic lethality as well as tumorigenesis [2]. These findings suggest that 53BP1 and BRCA1 battle each other to influence DSB repair pathway choice.Molecularly, 53BP1 is responsible for the defective HR seen in BRCA1-deficient cells. Furthermore, in those cells, 53BP1 promotes the formation of characteristic radial chromosomes that are caused by toxic NHEJ events, presumably during S phase. Understanding exactly how 53BP1 carries out its many functions has been a major challenge to the field as 53BP1 does not harbour any enzymatic activity. However, it has been shown that 53BP1 must accumulate on chromatin to be functional. In addition, a mutant 53BP1 allele in which all 28 ataxia telangiectasia-mutated (ATM) phosphorylation sites were changed to alanine (53BP1^28A) failed to rescue 53BP1 deficiency, suggesting that 53BP1 acts through phosphorylation-dependent protein interactions to promote NHEJ [2].RIF1 was identified as the first effector of 53BP1 in DSB repair [3,4,5,6,7]. RIF1 accumulates at DSB sites by binding to phosphorylated 53BP1 but, intriguingly, the loss of RIF1 has a milder effect than the loss of 53BP1 with respect to the fusion of dysfunctional telomeres [3], and RIF1 deficiency does not fully restore HR in BRCA1-deficient cells [7]. As the 53BP1^28A mutant is nearly as defective as the complete loss of 53BP1 for these activities, these observations indicate that additional 53BP1 effector proteins contribute to some of the 53BP1 functions.Nussenzweig and colleagues provide compelling evidence that the BRCT domain-containing protein PTIP is the missing 53BP1 effector protein [1]. The authors identified a separation-of-function mutation in 53BP1 that disrupted the first eight amino-terminal ATM sites (53BP1^8A). The 53BP1^8A mutant behaved the same as the wild-type protein with respect to CSR—a physiological process dependent on NHEJ—but failed to promote genome instability (radial chromosome formation) in BRCA1-deficient cells after treatment with a PARP inhibitor. Since RIF1-deficient cells have impaired CSR and RIF1 can localize to break sites in cells expressing the 53BP1^8A mutant, this suggests that a protein other than RIF1 binds to the N-terminal region of 53BP1 to inhibit HR.The newly identified 53BP1 effector protein PTIP is a multifunctional DNA repair factor that interacts with phosphorylated Ser 25 of 53BP1 through its tandem BRCT domains [8]—a site that was mutated in the 53BP1^8A allele. PTIP is also part of the MLL3/MLL4 histone H3 Lys 4 methyltransferase complexes but this function seems to be unrelated to its role as a 53BP1 co-factor.Nussenzweig and co-workers found that PTIP-deficient cells are sensitive to ionizing radiation but tolerant of DNA damaging agents that are toxic to HR-deficient cells, which suggests a role for PTIP in NHEJ. In agreement with this, the fusion frequency of uncapped telomeres was reduced in PTIP-deficient cells. Interestingly, as in the case of the 53BP1^8A allele, PTIP-deficient B cells were proficient in switching their immunoglobulin locus, although this switching event is impaired in RIF1^−/− B cells. This suggests that PTIP might participate selectively in pathological NHEJ.Nussenzweig and colleagues next generated a conditional BRCA1^−/− PTIP^−/− mouse to investigate the contribution of PTIP to the genome instability of BRCA1-deficient B cells. Loss of PTIP restored normal growth kinetics and genome stability to BRCA1-deficient cells treated with a PARP inhibitor. In addition, RAD51 IR-induced focus formation was restored in BRCA1^−/− PTIP^−/− cells. As the primary defect of BRCA1-deficient cells with respect to HR seems to be at the level of resection, the accumulation of the single-stranded DNA-binding protein RPA into IR-induced foci was then analysed. The finding that PTIP-deficient cells have an increased number of RPA foci per cell supports a role for PTIP in blocking resection. Together, this suggests that PTIP opposes DNA end resection and mutagenic DSB repair in BRCA1-deficient cells.These results were surprising as they revealed that the 53BP1 activities relating to physiological NHEJ (during CSR) and mutagenic NHEJ (after PARP inhibition) can be separated, and that they are carried out by two distinct proteins that ‘read'' ATM-dependent 53BP1 phosphorylation. The relationship between 53BP1, RIF1 and PTIP is probably complex, as suggested by the possible competition between RIF1 and PTIP, and the observation that both proteins contribute in an additive manner to the fusion of dysfunctional telomeres, downstream from 53BP1.According to these findings, multiple phosphorylation events in 53BP1 seem to integrate ATM activity to control distinct aspects of DSB repair pathway choice (Fig 1). Establishing exactly how an increase of ATM activity at break sites is translated into the coordination of 53BP1 phosphorylation, with RIF1 and PTIP binding, will be an important milestone towards understanding 53BP1 function. Indeed, multi-site phosphorylation and its recognition by binding proteins can be used to develop switch-like responses that might be important for organizing the chromatin at DSB sites.Open in a separate windowFigure 153BP1 phospho-dependent interactions involved in DSB repair. PTIP and RIF1 interact with chromatin-bound and ATM-phosphorylated 53BP1 at DSB sites. PTIP binds directly to 53BP1 phosphorylated on Ser 14;25 (within the first eight Ser/Thr-Q sites). RIF1 binds to phosphorylated 53BP1 either directly or through an intermediate factor (X). The carboxy-terminal seven Ser/Thr-Q sites (9–15 Ser/Thr-Q sites) are involved in the interaction of RIF1–53BP1, although the amino-terminal eight Ser/Thr-Q sites might stabilize the binding. It is unknown whether PTIP and RIF1 can associate simultaneously with 53BP1 (left side of the figure), or if the binding is exclusive, due to either differential phosphorylation of the Ser/Thr-Q sites or steric hindrance (right side of the figure). 53BP1, PTIP and RIF1 block DNA end-resection and promote NHEJ repair. Although both PTIP and RIF1 contribute to dysfunctional telomere fusions, they also have distinct functions downstream from 53BP1. While RIF1 is essential for CSR and has a milder effect on toxic NHEJ events, PTIP is dispensable for CSR and has a more prominent role in toxic NHEJ events that lead to genome instability in BRCA1-deficient cells. ATM, ataxia telangiectasia-mutated; CSR, class switch recombination; DSB, double-stranded break; NHEJ, non-homologous end-joining.The identification of PTIP as a new 53BP1 effector also deepens the mystery of DSB repair pathway choice regulation by 53BP1. Future studies are needed to elucidate how 53BP1 and its effector proteins block resection. Are PTIP and RIF1 blocking specific nucleases? Do they act in a temporally distinct fashion or are they distributed in distinct subdomains of the chromatin flanking DSB sites? What is the function of PTIP in relation to the cell cycle? Testing whether RIF1 binds directly to 53BP1, and if so to which phosphorylated site, might answer some of the above questions. The identification of a RIF1 mutation that selectively disrupts 53BP1 binding would enable surgical manipulation of the 53BP1–RIF1–PTIP circuit at DSB sites.Another unresolved issue is whether 53BP1 acts solely by recruiting RIF1 and PTIP, or whether 53BP1 has a more active role in blocking resection. We have shown that 53BP1 localizes to the chromatin flanking the DSBs by binding to methylated and ubiquitinated nucleosomes, in a wheel clamp-like manner [9]. This suggests that 53BP1 might modify the nucleosomal array structure in a way that makes it refractory to the resection machinery. Recognizing how nucleosomes modified by 53BP1 cooperate with RIF1 and PTIP might provide clues to the role of these two proteins in end protection.It is important to note that in human cells, PTIP might not be recruited to DSB sites in a 53BP1- and ATM-dependent manner [8]. Furthermore, in the avian B-cell line DT40, PTIP promotes HR instead of inhibiting it [10]. It will be important to revisit these studies to tease out whether these differences are due to context-, experiment- or species-specific effects.The identification of PTIP as a candidate genetic modifier of BRCA1-deficient tumours is an important finding. As noted by the authors, disabling the PTIP–53BP1 interaction pharmacologically might selectively restore HR in BRCA1-deficient cells, which might be useful in certain contexts, for example as a chemopreventive strategy.     

FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2

The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.     

USP28 Is Recruited to Sites of DNA Damage by the Tandem BRCT Domains of 53BP1 but Plays a Minor Role in Double-Strand Break Metabolism

The DNA damage response (DDR) is critical for genome stability and the suppression of a wide variety of human malignancies, including neurodevelopmental disorders, immunodeficiency, and cancer. In addition, the efficacy of many chemotherapeutic strategies is dictated by the status of the DDR. Ubiquitin-specific protease 28 (USP28) was reported to govern the stability of multiple factors that are critical for diverse aspects of the DDR. Here, we examined the effects of USP28 depletion on the DDR in cells and in vivo. We found that USP28 is recruited to double-strand breaks in a manner that requires the tandem BRCT domains of the DDR protein 53BP1. However, we observed only minor DDR defects in USP28-depleted cells, and mice lacking USP28 showed normal longevity, immunological development, and radiation responses. Our results thus indicate that USP28 is not a critical factor in double-strand break metabolism and is unlikely to be an attractive target for therapeutic intervention aimed at chemotherapy sensitization.     

Recruitment Kinetics of DNA Repair Proteins Mdc1 and Rad52 but Not 53BP1 Depend on Damage Complexity

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET  = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T₀ after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ₁. Mdc1 accumulates faster (T₀ = 17±2 s, τ₁ = 98±11 s) than 53BP1 (T₀ = 77±7 s, τ₁ = 310±60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T₀ = 73±16 s, τ₁ = 1050±270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ₁ of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.     

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G₁ phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G₁ to homologous recombination in S/G₂.     

The in vivo dynamic interplay of MDC1 and 53BP1 at DNA damage-induced nuclear foci

MDC1 (NFBD1) and 53BP1 are critical mediators of the mammalian DNA damage response (DDR) at nuclear foci. Here we show by quantitative imaging assays that MDC1 and 53BP1 are similar in total copy number (~1200 copies per focus), but differ substantially in dynamics at both replication-associated nuclear bodies in normal cells and DNA repair foci in ionizing radiation (IR)-damaged cells. The majority of MDC1 (~80%) is extremely mobile and under continuous exchange, with only a small fraction (~20%) remaining immobile at foci irrespective of IR treatment. By contrast, 53BP1 has a smaller mobile fraction (~35%) and a larger immobile fraction (~65%) at nuclear bodies, and becomes more dynamic (~20% increase in mobile pool) upon IR-induced DNA damage. More specifically, the dynamics of 53BP1 is dependent on a minimal foci-targeting region (1231-1709), and differentially regulated by its N-terminus (1-1231) and C-terminal tBRCT domain (1709-1972). Furthermore, MDC1 knockdown, or disruption of 53BP1-MDC1 interaction, reduced the number of 53BP1 molecules at foci by ~60%, but only modestly affected 53BP1 retention. This novel in vivo evidence reveals distinct dynamics of MDC1 and 53BP1 at different types of nuclear structures, and shows that MDC1 directly recruits and retains a subset of 53BP1 for DNA repair.     

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.     

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few ?-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and is the site of methylation by PRMT1.     

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.     

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double‐strand breaks (DSBs), but only in the G1‐phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1–2 and 7–8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1–2 and 4–5. The BRCT domains 4–5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S‐phase, we observed a checkpoint defect after siRNA‐mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.     

Mediator of DNA Damage Checkpoint 1 (MDC1) Regulates Mitotic Progression

Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053–32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.     

Role for 53BP1 Tudor domain recognition of p53 dimethylated at lysine 382 in DNA damage signaling

Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.     

A Non-catalytic Function of SETD1A Regulates Cyclin K and the DNA Damage Response

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AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation

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