DNA structure checkpoint pathways in Schizosaccharomyces pombe

The response to DNA damage includes a delay to progression through the cell cycle to aid DNA repair. Incorrectly replicated chromosomes (replication checkpoint) or DNA damage (DNA damage checkpoint) delay the onset of mitosis. These checkpoint pathways detect DNA perturbations and generate a signal. The signal is amplified and transmitted to the cell cycle machinery. Since the checkpoint pathways are essential for genome stability, the related proteins which are found in all eukaryotes (from yeast to mammals) are expected to have similar functions to the yeast progenitors. This review article focuses on the function of checkpoint proteins in the model system Schizosaccharomyces pombe. Checkpoint controls in Saccharomyces cerevisiae and mammalian cells are mentioned briefly to underscore common or diverse features.     

Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway

Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Deltaddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Deltaddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Deltaddb1 strain. The results suggest that many of the defects observed in Deltaddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.     

Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.     

Schizosaccharomyces pombe checkpoint response to DNA interstrand cross-links

Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the "checkpoint Rad" family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G(2) arrest following ICL induction. In cells able to bypass the G(2) block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G(2) arrest-deficient mutants. This likely reflects the fraction of cells escaping the G(2) damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.     

DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

Eukaryotic chromosome ends are protected from illicit DNA joining by protein-DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.     

Cooperative binding of single-stranded telomeric DNA by the Pot1 protein of Schizosaccharomyces pombe

The fission yeast Pot1 (protection of telomeres) protein is a single-stranded telomeric DNA-binding protein and is required to protect the ends of chromosomes. Its N-terminal DNA-binding domain, Pot1pN, shows sequence similarity to the first OB fold of the telomere-binding protein alpha subunit of Oxytricha nova. The minimal-length telomeric ssDNA required to bind Pot1pN was determined to consist of six nucleotides, GGTTAC, by gel filtration chromatography and filter-binding assay (K(D) = 83 nM). Pot1pN is a monomer, and each monomer binds one hexanucleotide. Experiments with nucleotide substitutions demonstrated that the central four nucleotides are crucial for binding. The dependence of Pot1pN-ssDNA binding on salt concentration was consistent with a single ionic contact between the protein and the ssDNA phosphate backbone, such that at physiological salt condition 83% of the free energy of binding is nonelectrostatic. Subsequent binding experiments with longer ssDNAs indicated that Pot1pN binds to telomeric ssDNA with 3' end preference and in a highly cooperative manner that mainly results from DNA-induced protein-protein interactions. Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.     

Recombinant expression and characterization of yeast Mrc1, a DNA replication checkpoint mediator

Abstract

In Saccharomyces cerevisiae, Mrc1 (homolog of human Claspin and mediator of replication checkpoint) is not only a part of the replication machine, but also participates in the replication stress response when DNA replication is blocked by hydroxyurea. Since Mrc1 is expressed in a small amount in cells and has many proteins interacting with it as a mediator, it is difficult to obtain Mrc1 with high concentration and purity. This article reports the purification of a stable truncation of Mrc1 and the full length Mrc1. High concentration and high purity of Mrc1 was obtained and the three-dimensional structure of Mrc1 was analyzed, which is a ring with a hole in the center. At the same time, we found that Mrc1 has an interaction with Rad24-RFC a clamp loader in the replication checkpoint, and can form a complex with it, implying that we can assemble large replication checkpoint complexes in vitro. These results initially reveal the ring structure of Mrc1 and its interaction with Rad24-RFC in replication checkpoints in S. cerevisiae.     

Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint

Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase epsilon, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding, but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HU. The bimodal Mrc1/Pol2 interaction may be an additional step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.     

Non-productive DNA damage binding by DNA glycosylase-like protein Mag2 from Schizosaccharomyces pombe

Schizosaccharomyces pombe contains two paralogous proteins, Mag1 and Mag2, related to the helix-hairpin-helix (HhH) superfamily of alkylpurine DNA glycosylases from yeast and bacteria. Phylogenetic analysis of related proteins from four Schizosaccharomyces and other fungal species shows that the Mag1/Mag2 duplication is unique to the genus Schizosaccharomyces and most likely occurred in its ancestor. Mag1 excises N3- and N7-alkylguanines and 1,N⁶-ethenoadenine from DNA, whereas Mag2 has been reported to have no detectible alkylpurine base excision activity despite high sequence and active site similarity to Mag1. To understand this discrepancy we determined the crystal structure of Mag2 bound to abasic DNA and compared it to our previously determined Mag1–DNA structure. In contrast to Mag1, Mag2 does not flip the abasic moiety into the active site or stabilize the DNA strand 5′ to the lesion, suggesting that it is incapable of forming a catalytically competent protein–DNA complex. Subtle differences in Mag1 and Mag2 interactions with the DNA duplex illustrate how Mag2 can stall at damage sites without fully engaging the lesion. We tested our structural predictions by mutational analysis of base excision and found a single amino acid responsible at least in part for Mag2's lack of activity. Substitution of Mag2 Asp56, which caps the helix at the base of the DNA intercalation loop, with the corresponding serine residue in Mag1 endows Mag2 with ?A excision activity comparable to Mag1. This work provides novel insight into the chemical and physical determinants by which the HhH glycosylases engage DNA in a catalytically productive manner.     

DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe.

We have tested mutants corresponding to 20 DNA repair genes of the fission yeast Schizosaccharomyces pombe for their ability to arrest in G2 after DNA damage. Of the mutants tested, four are profoundly defective in this damage dependent G2 arrest. In addition, these four mutants are highly sensitive to a transient inhibition of DNA synthesis by hydroxyurea. This suggests that the pathway responsible for the recognition of DNA damage and the subsequent mitotic arrest, shares many functions with the mechanism that controls the dependency of mitosis on the completion of S phase. The phenotype of these checkpoint rad mutants in wee mutant backgrounds indicate that the G2 arrest response is mediated either through, or in parallel with, the activity of the cdc2 gene product.     

Sap1 promotes the association of the replication fork protection complex with chromatin and is involved in the replication checkpoint in Schizosaccharomyces pombe

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.     

Site-specific ORC binding,pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins

Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Delta3 site, weakly to the Delta6 site and not at all to the remaining sequences. In situ, the footprint over Delta3 is extended during G(1) phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Delta2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre-replication complex and initiates DNA synthesis, plus an additional novel origin element (Delta9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa.     

Tel2 is required for activation of the Mrc1-mediated replication checkpoint

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Delta strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Delta cells especially at S phase. In contrast, the cds1Deltaswi1Delta mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.     

Mms22 preserves genomic integrity during DNA replication in Schizosaccharomyces pombe

The faithful replication of the genome, coupled with the accurate repair of DNA damage, is essential for the maintenance of chromosomal integrity. The MMS22 gene of Saccharomyces cerevisiae plays an important but poorly understood role in preservation of genome integrity. Here we describe a novel gene in Schizosaccharomyces pombe that we propose is a highly diverged ortholog of MMS22. Fission yeast Mms22 functions in the recovery from replication-associated DNA damage. Loss of Mms22 results in the accumulation of spontaneous DNA damage in the S- and G2-phases of the cell cycle and elevated genomic instability. There are severe synthetic interactions involving mms22 and most of the homologous recombination proteins but not the structure-specific endonuclease Mus81-Eme1, which is required for survival of broken replication forks. Mms22 forms spontaneous nuclear foci and colocalizes with Rad22 in cells treated with camptothecin, suggesting that it has a direct role in repair of broken replication forks. Moreover, genetic interactions with components of the DNA replication fork suggest that Mms2 functions in the coordination of DNA synthesis following damage. We propose that Mms22 functions directly at the replication fork to maintain genomic integrity in a pathway involving Mus81-Eme1.     

A novel DNA damage recognition protein in Schizosaccharomyces pombe

Toxic and mutagenic O⁶-alkylguanine adducts in DNA are repaired by O⁶-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1⁺, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O⁶-methylguanine in [³H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O⁶-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O⁶-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O⁶-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.     

Isolation and characterization of the Schizosaccharomyces pombe rhp9 gene: a gene required for the DNA damage checkpoint but not the replication checkpoint.

Checkpoint controls exist in eukaryotic cells to ensure that cells do not enter mitosis in the presence of DNA damage or unreplicated chromosomes. In Schizosaccharomyces pombe many of the checkpoint genes analysed to date are required for both the DNA damage and the replication checkpoints, an exception being chk1 . We report here on the characterization of nine new methylmethane sulphonate (MMS)-sensitive S.pombe mutants, one of which is defective in the DNA damage checkpoint but not the replication checkpoint. We have cloned and sequenced the corresponding gene. The predicted protein is most similar to the Saccharomyces cerevisiae Rad9 protein, having 46% similarity and 26% identity. The S.pombe protein, which we have named Rhp9 (Rad9 homologue in S. pombe) on the basis of structural and phenotypic similarity, also contains motifs present in BRCA1 and 53BP1. Deletion of the gene is not lethal and results in a DNA damage checkpoint defect. Epistasis analysis with other S.pombe checkpoint mutants indicates that rhp9 acts in a process involving the checkpoint rad genes and that the rhp9 mutant is phenotypically very similar to chk1.     

Solution structure of the Ras binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe.

BACKGROUND: After activation, small GTPases such as Ras transfer the incoming signal to effectors by specifically interacting with the binding domain of these proteins. Structural details of the binding domain of different effectors determine which pathway is predominantly activated. Byr2 from fission yeast is a functional homolog of Raf, which is the direct downstream target of Ras in mammalians that initiates a protein kinase cascade. The amino acid sequence of Byr2's Ras binding domain is only weakly related to that of Raf, and Byr2's three-dimensional structure is unknown. RESULTS: We have solved the 3D structure of the Ras binding domain of Byr2 (Byr2RBD) from Schizosaccharomyces pombe in solution. The structure consists of three alpha helices and a mixed five-stranded beta pleated sheet arranged in the topology betabetaalphabetabetaalphabetaalpha with the first seven canonic secondary structure elements forming a ubiquitin superfold. 15N-(1)H-TROSY-HSQC spectroscopy of the complex of Byr2RBD with Ras*Mg(2+)*GppNHp reveals that the first and second beta strands and the first alpha helix of Byr2 are mainly involved in the protein-protein interaction as observed in other Ras binding domains. Although the putative interaction site of H-Ras from human and Ras1 from S. pombe are identical in sequence, binding to Byr2 leads to small but significant differences in the NMR spectra, indicating a slightly different binding mode. CONCLUSIONS: The ubiquitin superfold appears to be the general structural motif for Ras binding domains even in cases with vanishing sequence identity. However, details of the 3D structure and the interacting interface are different, thereby determining the specifity of the recognition of Ras and Ras-related proteins.