Checkpoint Protein Crb2 Research Articles

Hyperactive Cdc2 kinase interferes with the response to broken replication forks by trapping S.pombe Crb2 in its mitotic T215 phosphorylated state.

Although it is well established that Cdc2 kinase phosphorylates the DNA damage checkpoint protein Crb253BP1 in mitosis, the full impact of this modification is still unclear. The Tudor-BRCT domain protein Crb2 binds to modified histones at DNA lesions to mediate the activation of Chk1 by Rad3ATR kinase. We demonstrate here that fission yeast cells harbouring a hyperactive Cdc2CDK1 mutation (cdc2.1w) are specifically sensitive to the topoisomerase 1 inhibitor camptothecin (CPT) which breaks DNA replication forks. Unlike wild-type cells, which delay only briefly in CPT medium by activating Chk1 kinase, cdc2.1w cells bypass Chk1 to enter an extended cell-cycle arrest which depends on Cds1 kinase. Intriguingly, the ability to bypass Chk1 requires the mitotic Cdc2 phosphorylation site Crb2-T215. This implies that the presence of the mitotic phosphorylation at Crb2-T215 channels Rad3 activity towards Cds1 instead of Chk1 when forks break in S phase. We also provide evidence that hyperactive Cdc2.1w locks cells in a G1-like DNA repair mode which favours non-homologous end joining over interchromosomal recombination. Taken together, our data support a model such that elevated Cdc2 activity delays the transition of Crb2 from its G1 to its G2 mode by blocking Srs2 DNA helicase and Casein Kinase 1 (Hhp1).

Phosphorylation-Dependent Interactions between Crb2 and Chk1 Are Essential for DNA Damage Checkpoint

In response to DNA damage, the eukaryotic genome surveillance system activates a checkpoint kinase cascade. In the fission yeast Schizosaccharomyces pombe, checkpoint protein Crb2 is essential for DNA damage-induced activation of downstream effector kinase Chk1. The mechanism by which Crb2 mediates Chk1 activation is unknown. Here, we show that Crb2 recruits Chk1 to double-strand breaks (DSBs) through a direct physical interaction. A pair of conserved SQ/TQ motifs in Crb2, which are consensus phosphorylation sites of upstream kinase Rad3, is required for Chk1 recruitment and activation. Mutating both of these motifs renders Crb2 defective in activating Chk1. Tethering Crb2 and Chk1 together can rescue the SQ/TQ mutations, suggesting that the main function of these phosphorylation sites is promoting interactions between Crb2 and Chk1. A 19-amino-acid peptide containing these SQ/TQ motifs is sufficient for Chk1 binding in vitro when one of the motifs is phosphorylated. Remarkably, the same peptide, when tethered to DSBs by fusing with either recombination protein Rad22/Rad52 or multi-functional scaffolding protein Rad4/Cut5, can rescue the checkpoint defect of crb2Δ. The Rad22 fusion can even bypass the need for Rad9-Rad1-Hus1 (9-1-1) complex in checkpoint activation. These results suggest that the main role of Crb2 and 9-1-1 in DNA damage checkpoint signaling is recruiting Chk1 to sites of DNA lesions.

A new method to efficiently induce a site‐specific double‐strand break in the fission yeast Schizosaccharomyces pombe

Double-strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double-strand break into the genome by regulating the expression of the site-specific HO endonuclease with a galactose inducible promoter. Variations of the HO site-specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I-PpoI is tightly regulated with a tetracycline-inducible promoter. We show that induction of the I-PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break-adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well-conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double-stranded DNA breaks.

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.

Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand breaks

Cellular responses to DNA damage involve the relocalization of checkpoint proteins to DNA double-strand breaks (DSBs). The fission yeast checkpoint mediator protein Crb2, a homolog of mammalian 53BP1, forms ionizing radiation-induced nuclear foci (IRIF). The IRIF formation by Crb2 requires histone H2A C-terminal phosphorylation and H4-K20 methylation. However, the relevance of Crb2 relocalization is uncertain, because neither histone modification is required for a checkpoint response. Here we show that these histone modifications cooperate in the same Crb2 recruitment pathway, which also requires the Tudor and BRCT motifs in Crb2. In the absence of these histone modifications, an alternative recruitment pathway is sufficient for checkpoint activation and accumulation of Crb2 at a persistent DSB generated by HO endonuclease. This parallel pathway requires a cyclin-dependent kinase phosphorylation site in Crb2 that mediates an association with another BRCT protein Cut5 (the TopBP1 homolog), which also accumulates at HO-induced DSBs. We propose that such dual recruitment mechanisms may be a common feature of DNA damage checkpoint mediators.

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.

Methylation of Histone H4 Lysine 20 Controls Recruitment of Crb2 to Sites of DNA Damage

Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, we find the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a “histone mark” required for the recruitment of the checkpoint protein Crb2.

Homo-oligomerization Is the Essential Function of the Tandem BRCT Domains in the Checkpoint Protein Crb2

BRCT (BRCA1 C terminus) domains are frequently found as a tandem repeat in proteins involved in DNA damage responses, such as Saccharomyces cerevisiae Rad9, human 53BP1 and BRCA1. Tandem BRCT domains mediate protein-protein and protein-DNA interactions. However, the functional significance of these interactions is largely unknown. Here we report the oligomerization of Schizosaccharomyces pombe checkpoint protein Crb2 through its tandem BRCT domains. Truncated Crb2 without BRCT domains is defective in DNA damage checkpoint signaling. However, addition of either of two heterologous dimerization motifs largely restores the functions of truncated Crb2 without BRCT domains. Replacement of Crb2 BRCT domains with a dimerization motif also renders cells resistant to the dominant negative effect of overexpressing Crb2 BRCT domains. These results demonstrate that the crucial function of the tandem BRCT domains is to oligomerize Crb2.

Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes.

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.

Cdc2-cyclin B kinase activity links Crb2 and Rqh1-topoisomerase III.

The availability of a sister chromatid, and thus the cell cycle phase in which DNA double-strand breaks (DSBs) occur, influences the choice between homologous recombination (HR) or nonhomologous end joining (NHEJ). The sequential activation and destruction of CDK-cyclin activities controls progression through the cell cycle. Here we provide evidence that the major Schizosaccharomyces pombe CDK, Cdc2-cyclin B, influences recombinational repair of radiation-induced DSBs during the G(2) phase at two distinct stages. At an early stage in HR, a defect in Cdc2 kinase activity, which is caused by a single amino acid change in cyclin B, affects the formation of Rhp51 (Rad51(sp)) foci in response to ionizing radiation in a process that is redundant with the function of Rad50. At a late stage in HR, low Cdc2-cyclin B activity prevents the proper regulation of topoisomerase III (Top3) function, disrupting a recombination step that occurs after the assembly of Rhp51 foci. This effect of Cdc2-cyclin B kinase on Top3 function is mediated by the BRCT-domain-containing checkpoint protein Crb2, thus linking checkpoint proteins directly with recombinational repair in G(2). Our data suggest a model in which CDK activity links processing of recombination intermediates to cell cycle progression via checkpoint proteins.

Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1.

Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.