Cooperative control of Crb2 by ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast

The cellular responses to double-stranded breaks (DSBs) typically involve the extensive accumulation of checkpoint proteins in chromatin surrounding the damaged DNA. One well-characterized example involves the checkpoint protein Crb2 in the fission yeast Schizosaccharomyces pombe. The accumulation of Crb2 at DSBs requires the C-terminal phosphorylation of histone H2A (known as gamma-H2A) by ATM family kinases in chromatin surrounding the break. It also requires the constitutive methylation of histone H4 on lysine-20 (K20). Interestingly, neither type of histone modification is essential for the Crb2-dependent checkpoint response. However, H4-K20 methylation is essential in a crb2-T215A strain that lacks a cyclin-dependent kinase phosphorylation site in Crb2. Here we explain this genetic interaction by describing a previously overlooked effect of the crb2-T215A mutation. We show that crb2-T215A cells are able to initiate but not sustain a checkpoint response. We also report that gamma-H2A is essential for the DNA damage checkpoint in crb2-T215A cells. Importantly, we show that inactivation of Cdc2 in gamma-H2A-defective cells impairs Crb2-dependent signaling to the checkpoint kinase Chk1. These findings demonstrate that full Crb2 activity requires phosphorylation of threonine-215 by Cdc2. This regulation of Crb2 is independent of the histone modifications that are required for the hyperaccumulation of Crb2 at DSBs.     

Di-methyl H4 lysine 20 targets the checkpoint protein Crb2 to sites of DNA damage

Histone lysine methylation is an important chromatin modification that can be catalyzed to a mono-, di-, or tri-methyl state. An ongoing challenge is to decipher how these different methyllysine histone marks can mediate distinct aspects of chromatin function. The fission yeast checkpoint protein Crb2 is rapidly targeted to sites of DNA damage after genomic insult, and this recruitment requires methylation of histone H4 lysine 20 (H4K20). Here we show that the tandem tudor domains of Crb2 preferentially bind the di-methylated H4K20 residue. Loss of this interaction by disrupting either the tudor-binding motif or the H4K20 methylating enzyme Set9/Kmt5 ablates Crb2 localization to double-strand breaks and impairs checkpoint function. Further we show that dimethylation, but not tri-methylation, of H4K20 is required for Crb2 localization, checkpoint function, and cell survival after DNA damage. These results argue that the di-methyl H4K20 modification serves as a binding target that directs Crb2 to sites of genomic lesions and defines an important genome integrity pathway mediated by a specific methyl-lysine histone mark.     

Regulation of checkpoint kinases through dynamic interaction with Crb2

ATR/Rad3-like kinases promote the DNA damage checkpoint through regulating Chk1 that restrains the activation of cyclin-dependent kinases. In fission yeast, Crb2, a BRCT-domain protein that is similar to vertebrate 53BP1, plays a crucial role in establishing this checkpoint. We report here that Crb2 regulates DNA damage checkpoint through temporal and dynamic interactions with Rad3, Chk1 and replication factor Cut5. The active complex formation between Chk1 and Crb2 is regulated by Rad3 and became maximal during the checkpoint arrest. Chk1 activation seems to need two steps of interaction changes: the loss of Rad3-Chk1 and Rad3-Crb2 interactions, and the association between hyperphosphorylated forms of Chk1 and Crb2. Chk1 is the major checkpoint kinase for the arrest of DNA polymerase mutants. The in vitro assay of Chk1 showed that its activation requires the presence of Crb2 BRCT. Hyperphosphorylation of Crb2 is also dependent on its intact BRCT. Finally, we show direct interaction between Rad3 and Crb2, which is inhibitory to Rad3 activity. Hence, Crb2 is the first to interact with both Rad3 and Chk1 kinases.     

Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint.

DNA damage induces cell cycle arrest (called the damage checkpoint), during which cells carry out actions for repair. A fission yeast protein, Crb2/Rhp9, which resembles budding yeast Rad9p and human BRCA1, promotes checkpoint by activating Chk1 kinase, which restrains Cdc2 activation. We show here that phosphorylation of the T215 Cdc2 site of Crb2 is required for reentering the cell cycle after the damage-induced checkpoint arrest. If this site is nonphosphorylatable, irradiated cells remain arrested, though damage is repaired, and maintain the phosphorylated state of Chk1 kinase. The T215 site is in vitro phosphorylated by purified Cdc2 kinase. Phosphorylation of T215 occurs intensely in response to DNA damage at a late stage, suggesting an antagonistic role of Cdc2 phosphorylation toward checkpoint.     

The tandem BRCT domain of 53BP1 is not required for its repair function

53BP1 plays an important role in cellular response to DNA damage. It is thought to be the mammalian homologue of budding yeast Rad9 and/or fission yeast Crb2. Rad9/Crb2 are bona fide checkpoint proteins whose activation requires their corresponding C-terminal tandem BRCT (BRCA1 C-terminal) motifs, which mediate their oligomerization and phosphorylation at multiple sites following DNA damage. Here we show that the function of human 53BP1 similarly depends on its oligomerization and phosphorylation at multiple sites but in a BRCT domain-independent manner. Moreover, unlike its proposed yeast counterparts, human 53BP1 only has limited checkpoint functions but rather acts as an adaptor in the repair of DNA double strand breaks. This difference in function may reflect the higher complexity of the DNA damage response network in metazoa including the evolution of other BRCT domain-containing proteins that may have functions redundant or overlapping with those of 53BP1.     

The tandem BRCT domains of Ect2 are required for both negative and positive regulation of Ect2 in cytokinesis

Epithelial cell transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases, molecular switches essential for the control of cytokinesis in mammalian cells. Aside from the canonical Dbl homology/pleckstrin homology cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains. In this study, we address the role of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis. First, we show that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, we provide evidence that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of an N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, the results of our study indicate that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. Whereas these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis.     

Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains

BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding.     

Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping

In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5′ strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.     

Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein

Protein interactions critical to DNA repair and cell cycle control systems are often coordinated by modules that belong to a superfamily of structurally conserved BRCT domains. Because the mechanisms of BRCT interactions and their significance are not well understood, we sought to define the affinity and specificity of those BRCT modules that orchestrate base excision repair and single-strand break repair. Common to these pathways is the essential XRCC1 DNA repair protein, which interacts with at least nine other proteins and DNA. Here, we characterized the interactions of four purified BRCT domains, two from XRCC1 and their two partners from DNA ligase IIIalpha and poly(ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase IIIalpha BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase IIIalpha BRCT complex. To examine the assembly states of isolated BRCT domains and pairwise domain complexes, we used size-exclusion chromatography coupled with on-line light scattering. This analysis indicated that isolated BRCT domains form homo-oligomers and that the BRCT complex between the C-terminal XRCC1 domain and the ligase IIIalpha domain is a heterotetramer with 2:2 stoichiometry. Using affinity capture and surface plasmon resonance methods, we determined that specific heteromeric interactions with high nanomolar dissociation constants occur between pairs of cognate BRCT domains. A structural model for a XRCC1 x DNA ligase IIIalpha heterotetramer is proposed as a core base excision repair complex, which constitutes a scaffold for higher order complexes to which other repair proteins and DNA are brought into proximity.     

Specific recognition of phosphorylated tail of H2AX by the tandem BRCT domains of MCPH1 revealed by complex structure

MCPH1 is especially important for linking chromatin remodeling to DNA damage response. It contains three BRCT (BRCA1-carboxyl terminal) domains. The N-terminal region directly binds with chromatin remodeling complex SWI-SNF, and the C-terminal BRCT2-BRCT3 domains (tandem BRCT domains) are involved in cellular DNA damage response. The MCPH1 gene associates with evolution of brain size, and its variation can cause primary microcephaly. In this study we solve the crystal structures of MCPH1 natural variant (A761) C-terminal tandem BRCT domains alone as well as in complex with γH2AX tail. Compared with other structures of tandem BRCT domains, the most significant differences lie in phosphopeptide binding pocket. Additionally, fluorescence polarization assays demonstrate that MCPH1 tandem BRCT domains show a binding selectivity on pSer +3 and prefer to bind phosphopeptide with free COOH-terminus. Taken together, our research provides new structural insights into BRCT-phosphopeptide recognition mechanism.     

The BRCT domains of ECT2 have distinct functions during cytokinesis

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Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background  

Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.     

Crystal structure of the BARD1 BRCT domains

The interaction of the breast tumor suppressor BRCA1 with the protein BARD1 results in the formation of a heterodimeric complex that has ubiquitin ligase activity and plays central roles in cell cycle checkpoint control and DNA repair. Both BRCA1 and BARD1 possess a pair of tandem BRCT domains that interact in a phosphorylation-dependent manner with target proteins. We determined the crystal structure of the human BARD1 BRCT repeats (residues 568-777) at 1.9 A resolution. The composition and structure of the BARD1 phosphoserine-binding pocket P1 are strikingly similar to those of the BRCA1 and MDC1 BRCT domains, suggesting a similar mode of interaction with the phosphate group of the ligand. By contrast, the BARD1 BRCT selectivity pocket P2 exhibits distinct structural features, including two prominent histidine residues, His685 and His686, which may be important for ligand binding. The protonation state of these histidines has a marked effect on the calculated electrostatic potential in the vicinity of P2, raising the possibility that ligand recognition may be regulated by changes in pH. Importantly, the BARD1 BRCT structure provides insights into the mechanisms by which the cancer-associated missense mutations C645R, V695L, and S761N may adversely affect the structure and function of BARD1.     

BRCT Domain Interactions with Phospho-Histone H2A Target Crb2 to Chromatin at Double-Strand Breaks and Maintain the DNA Damage Checkpoint

Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3^ATR, forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT₂) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1^ATM and Rad3^ATR, although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A (γ-H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT₂ domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT₂ γ-H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the γ-H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins.Double-strand breaks (DSBs) are among the most dangerous forms of DNA damage (26, 30). Human cells experience DSBs several times a day, either during normal metabolism or as a consequence of exposure to DNA-damaging agents, such as ionizing radiation (IR) (18). Importantly, the unfaithful repair of such breaks can result in genome instability and cancer. The response to DSBs is coordinated by a conserved signal transduction cascade, which leads to cell cycle arrest and activation of DNA repair and constitutes the checkpoint response (9, 14, 20). The essential players in this process fall into four groups: sensors, mediators, transducers, and effectors (20). Sensors are the first to recognize and bind to DNA breaks and include the Mre11-Rad50-Nbs1 complex in humans and Schizosaccharomyces pombe (Mre11-Rad50-Xrs2 in Saccharomyces cerevisiae). The PIKKs (phosphoinositide 3-kinase-like kinases) ATR-ATRIP (ScMec1-ScDdc2/SpRad3-SpRad26) and ATM (ScTel1/SpTel1) act as transducers that transmit the signal to the effector kinases Chk1 (ScChk1/SpChk1) and Chk2 (ScRad53/SpCds1), whose role is to target downstream targets, such as p53 in mammals, and to amplify the signal (9, 14, 20).Signaling between transducers and effectors is facilitated and enhanced by mediator proteins (19, 20). In the fission yeast Schizosaccharomyces pombe, Crb2/Rhp9 is a critical mediator of the DNA damage checkpoint (31, 42) and is related to Saccharomyces cerevisiae Rad9 and mammalian 53BP1 (p53 binding protein 1). Rad3^ATR-Rad26^ATRIP phosphorylates Crb2 in response to damage, and Crb2 is required for phosphorylation of Chk1 by Rad3^ATR-Rad26^ATRIP (31). Chk1, in turn, restrains entry into mitosis by phosphorylating and thus inactivating the phosphatase Cdc25 that is a mitotic inducer (10, 11, 28). Crb2-null cells are sensitive to a range of genotoxins and are unable to delay division in response to DNA damage (31, 42).Crb2 is a nuclear protein that rapidly relocalizes to DSBs. This occurs on such a large scale that IR-induced nuclear foci (IRIF) of yellow fluorescent protein (YFP)-tagged Crb2 expressed from the endogenous promoter are readily detected by live cell microscopy (5). These foci colocalize with homologous recombination (HR) repair factors such as Rad22^Rad52. Two types of histone modifications regulate Crb2 localization at DSBs: C-terminal phosphorylation of histone H2A, denoted as γ-H2A (23), and lysine-20 dimethylation of histone H4, denoted as H4-K20me2 (32). Phosphorylation of an SQ motif within the C-terminal tail of histone H2A of budding yeast or fission yeast, or the H2AX variant in mammals, is one of the earliest cellular responses triggered by DNA damage (3, 23, 29). The γ-H2A/X modification, which is catalyzed by the checkpoint kinases ATR^Rad3 and ATM^Tel1, spans large distances on both sides of a DSB, and it plays a critical role in recruiting DNA damage response proteins, chromatin remodeling complexes, and cohesin (2, 21, 23, 34, 35, 37, 38, 40). Protein crystallography and biochemical studies established that mammalian Mdc1, S. pombe Crb2, and Brc1 DNA damage response proteins directly bind the phosphorylated tail of histone H2A/X through tandem C-terminal BRCT domains (16, 35, 40). In contrast to γ-H2A, H4-K20 methylation catalyzed by Set9/Kmt5 histone methyltransferase appears to be constitutive and not regulated by DNA damage (32). H4-K20me2 directly binds tandem tudor domains (Tudor₂) located to the N-terminal side of the BRCT domains in Crb2 (1).YFP-Crb2 does not form IRIF in hta1-S129A hta2-S128A (htaAQ) or rad3Δ tel1Δ cells, in which γ-H2A phosphorylation is abolished (23), or in set9Δ cells or tudor domain mutants of Crb2 that ablate binding to H4-K20me2 (6, 32). However, Crb2 checkpoint functions are only partially impaired in an htaAQ set9Δ strain, implying that physiologically significant recruitment of Crb2 to DSBs also occurs by a histone modification-independent pathway. Indeed, we found that YFP-Crb2 forms microscopically visible foci in htaAQ set9Δ cells when DSBs are created by HO endonuclease or by treating cells in G₁ phase with IR (6). Unlike IR-induced DSBs formed during G₂ phase, these types of DSBs lack an intact sister chromatid that can be used for HR repair and therefore they are highly persistent. Further analysis revealed that the histone modification-independent pathway of recruiting Crb2 to DSBs requires threonine-215 (Thr215) phosphorylation catalyzed by the cyclin-dependent kinase (CDK) Cdc2, which facilitates an interaction with Cut5 (ScDpb11; mammalian TopBP1) (6, 8, 31). The crb2-T215A mutation does not ablate YFP-Crb2 IRIF formation; however, Crb2 Thr215 phosphorylation is required for formation of YFP-Crb2 foci at persistent DSBs in htaAQ or set9Δ cells, and combining crb2-T215A with htaAQ or set9Δ abolishes Crb2 function (6).The tandem C-terminal BRCT domains (BRCT₂) of Crb2 not only mediate interactions with γ-H2A but also coordinate Crb2 homodimerization (4). In fact, replacing BRCT₂ with a leucine zipper (LZ) dimerization motif restores substantial function to Crb2 without restoring its ability to form IRIF. Thus, the most crucial task of the Crb2 BRCT domains is to provide a homodimerization platform, while binding to γ-H2A provides an additional function that is necessary for full resistance to DNA damage (4).In a recent study, Kilkenny et al. (16) solved the crystal structures of Crb2-BRCT₂ alone and in complex with a γ-H2A-derived phosphopeptide containing the common C-terminal residues of H2A.1 and H2A.2 (the two H2A paralogues in S. pombe). These analyses revealed the structural determinants of BRCT₂ binding to γ-H2A and BRCT₂-mediated homodimerization of Crb2. Ser666 was found to be critical for homodimerization in vitro, and mutation of this residue severely impaired Crb2 function in vivo. Residues Ser548 and Lys619 were identified as important for the interaction with the phosphate group on γ-H2A.1 pSer129. However, a charge reversal mutation of Lys619 did not abrogate Crb2 IRIF formation measured using methanol-fixed cells, although it did disrupt binding to a γ-H2A peptide in vitro (16). These unexpected findings indicated that γ-H2A likely has an indirect role in regulating Crb2 localization at DSBs. Here, we investigate Crb2 localization in live cells and find that while mutations of Ser548 or Lys619 partially impair Crb2 IRIF, the corresponding double mutant is severely deficient in Crb2 IRIF formation. Our findings and an independent study by Sanders et al. (33) show that γ-H2A binding to BRCT₂ is critical for Crb2 focus formation at IR-induced DSBs and for maintaining a DNA damage checkpoint response.     

Regulation of AURORA B function by mitotic checkpoint protein MAD2

Cell cycle checkpoint signaling stringently regulates chromosome segregation during cell division. MAD2 is one of the key components of the spindle and mitotic checkpoint complex that regulates the fidelity of cell division along with MAD1, CDC20, BUBR1, BUB3 and MAD3. MAD2 ablation leads to erroneous attachment of kinetochore-spindle fibers and defective chromosome separation. A potential role for MAD2 in the regulation of events beyond the spindle and mitotic checkpoints is not clear. Together with active spindle assembly checkpoint signaling, AURORA B kinase activity is essential for chromosome condensation as cells enter mitosis. AURORA B phosphorylates histone H3 at serine 10 and serine 28 to facilitate the formation of condensed metaphase chromosomes. In the absence of functional AURORA B cells escape mitosis despite the presence of misaligned chromosomes. In this study we report that silencing of MAD2 results in a drastic reduction of metaphase-specific histone H3 phosphorylation at serine 10 and serine 28. We demonstrate that this is due to mislocalization of AURORA B in the absence of MAD2. Conversely, overexpression of MAD2 concentrated the localization of AURORA B at the metaphase plate and caused hyper-phosphorylation of histone H3. We find that MAD1 plays a minor role in influencing the MAD2-dependent regulation of AURORA B suggesting that the effects of MAD2 on AURORA B are independent of the spindle checkpoint complex. Our findings reveal that, in addition to its role in checkpoint signaling, MAD2 ensures chromosome stability through the regulation of AURORA B.