RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.     

Nucleolar exit of RNF8 and BRCA1 in response to DNA damage

The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to γ-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did γ-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage.     

The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks

Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.     

Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain

MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.     

DNA damage signaling in response to double-strand breaks during mitosis

The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a “primary” DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11–Rad50–Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.     

When cleavage is not attractive: non-catalytic inhibition of ubiquitin chains at DNA double-strand breaks by OTUB1

Non-degradative ubiquitylation plays a crucial role in many cellular signaling pathways, including the DNA damage response. Two ubiquitin ligases, RNF8 and RNF168, in combination with the E2 ubiquitin conjugating enzyme UBC13 catalyze the formation of K63-linked ubiquitin chains at sites of DNA double-strand breaks to promote their faithful repair. However, little is known about their negative regulation. A recent study identifies a deubiquitylating enzyme, OTUB1, which counteracts RNF8/RNF168-dependent ubiquitin chain formation at break sites. Surprisingly, this enzyme carries out its function not by cleavage of polyubiquitin chains, but by targeting UBC13. This non-canonical role for a deubiquitylating enzyme has implications for the regulation of ubiquitylation not just in DNA repair, but potentially in many other cellular signaling processes.     

FHA Domain-Mediated DNA Checkpoint Regulation of Rad53

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.     

Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans

The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.     

Noncanonical E2 variant-independent function of UBC13 in promoting checkpoint protein assembly

The E2 ubiquitin-conjugating enzyme UBC13 plays pivotal roles in diverse biological processes. Recent studies have elucidated that UBC13, in concert with the E3 ubiquitin ligase RNF8, propagates the DNA damage signal via a ubiquitylation-dependent signaling pathway. However, mechanistically how UBC13 mediates its role in promoting checkpoint protein assembly and its genetic requirement for E2 variants remain elusive. Here we provide evidence to support the idea that the E3 ubiquitin ligase complex RNF8-UBC13 functions independently of E2 variants and is sufficient in facilitating ubiquitin conjugations and accumulation of DNA damage mediator 53BP1 at DNA breaks. The RNF8 RING domain serves as the molecular platform to anchor UBC13 at the damaged chromatin, where localized ubiquitylation events allow sustained accumulation of checkpoint proteins. Intriguingly, we found that only a group of RING domains derived from E3 ubiquitin ligases, which have been shown to interact with UBC13, enabled UBC13-mediated FK2 and 53BP1 focus formation at DNA breaks. We propose that the RNF8 RING domain selects and loads a subset of UBC13 molecules, distinct from those that exist as heterodimers, onto sites of double-strand breaks, which facilitates the amplification of DNA damage signals.     

FHA domain-mediated DNA checkpoint regulation of Rad53

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain

Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.     

Phosphorylation of SMURF2 by ATM exerts a negative feedback control of DNA damage response

Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities. We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with RNF20 (ring finger protein 20) in shaping chromatin landscape and preserving genomic stability. However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and MS analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at Ser³⁸⁴ by ataxia telangiectasia mutated (ATM) serine/threonine kinase, and this phosphorylation is required for its interaction with RNF20. We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly. More importantly, mouse embryonic fibroblasts expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared with those expressing WT SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ataxia telangiectasia mutated–induced phosphorylation at Ser³⁸⁴ constitutes a negative feedback loop that regulates DSB repair.     

BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G(2)/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.     

RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins

Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.     

Nibrin forkhead-associated domain and breast cancer C-terminal domain are both required for nuclear focus formation and phosphorylation

The Mre11.Rad50.nibrin protein complex plays an essential role in the mammalian cellular response to DNA double-strand breaks. The disorder Nijmegen breakage syndrome (NBS) results from mutations in the NBS1 gene that encodes nibrin, and NBS cells are radiosensitive and defective in S-phase checkpoint activation following irradiation. In response to radiation, nibrin is phosphorylated by Atm, and the Mre11.Rad50.nibrin complex relocalizes to form punctate nuclear foci. The N terminus of nibrin contains a forkhead-associated (FHA) domain and a breast cancer C-terminal (BRCT) domain, the functions of which are unclear. To determine the role of the FHA and BRCT domains in nibrin function, we have performed site-directed mutagenesis of conserved residues in these motifs. Mutations in the nibrin FHA and BRCT domains did not affect interaction with Mre11.Rad50 or nuclear localization of the complex. However, mutation of conserved residues in either domain disrupted nuclear focus formation and blocked nibrin phosphorylation after irradiation, suggesting that these events may be functionally interdependent. Despite an effect on nibrin phosphorylation, expression of the FHA or BRCT mutants in NBS cells restored the downstream phosphorylation of Chk2 and Smc1, necessary for S-phase checkpoint activation. None of the mutations revealed separate functions for the FHA or BRCT domains, suggesting they do not function independently.     

DNA-damage checkpoints: location, location, location

The DNA-damage response (DDR) is an evolutionarily conserved signaling cascade crucial for sensing DNA damage and activating cellular responses such as cell-cycle arrest, DNA repair, senescence and apoptosis. Excitingly, two recent studies describe activation of this checkpoint in the absence of DNA damage. These studies support the idea that accumulation of checkpoint proteins and changes in global-chromatin structure are important signaling intermediates for the activation of the DDR.     

Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians

The maintenance of genomic stability relies on the coordinated action of a number of cellular processes, including activation of the DNA-damage checkpoint, DNA replication, DNA repair, and telomere homeostasis. Many proteins involved in these cellular processes use different types of functional modules to regulate and execute their functions. Recent studies have revealed that many DNA-damage checkpoint and DNA repair proteins in human cells possess the oligonucleotide/oligosaccharide-binding (OB) fold domains, which are known to bind single-stranded DNA in both prokaryotes and eukaryotes. Furthermore, during the DNA damage response, the OB folds of the human checkpoint and DNA repair proteins play critical roles in DNA binding, protein complex assembly, and regulating protein–protein interactions. These findings suggest that the OB fold is an evolutionarily conserved functional module that is widely used by genome guardians. In this review, we will highlight the functions of several well-characterized or newly discovered eukaryotic OB-fold proteins in the DNA damage response.