Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.

Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein–protein interactions.

Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsBrad3 and uvsDrad26 have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIMEcyclinB, but ectopic expression of active nondegradable NIMEcyclinB does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMXcdc2 and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

Roles for Basal and Stimulated p21Cip-1/WAF1/MDA6 Expression and Mitogen-activated Protein Kinase Signaling in Radiation-induced Cell Cycle Checkpoint Control in Carcinoma Cells

We investigated the role of the cdk inhibitor protein p21Cip-1/WAF1/MDA6 (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0–10 min) and secondary (90–240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G1 that were p21 and MAPK dependent, whereas the elevation of cells present in G2/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G2/M phase 24–48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G2/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G2/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G2/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G1/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G2/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.

p53 regulates a G2 checkpoint through cyclin B1

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, we have derived a human ovarian cell that undergoes p53-dependent G2 arrest at 32°C. We have found that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation.

Role of Fission Yeast Primase Catalytic Subunit in the Replication Checkpoint

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1+ and spp2+ (S. pombe primase 1 and 2). spp1+ encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polα to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1+ gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1+ exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1+ reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polα–primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polα has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polα. Together, these have led us to propose that suboptimal cellular levels of polα–primase complex due to the allele-specific mutations of Spp1 might not allow Polα to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.

Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe.

All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.

cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein.

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.

Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls.

The human hepatitis B virus (HBV) HBx protein is a small transcriptional activator that is essential for virus infection. HBx is thought to be involved in viral hepatocarcinogenesis because it promotes tumorigenesis in transgenic mice. HBx activates the RAS-RAF-mitogen-activated protein (MAP) kinase signaling cascade, through which it activates transcription factors AP-1 and NF-kappa B, and stimulates cell DNA synthesis. We show that HBx stimulates cell cycle progression, shortening the emergence of cells from quiescence (G0) and entry into S phase by at least 12 h, and accelerating transit through checkpoint controls at G0/G1 and G2/M. Compared with serum stimulation, HBx was found to strongly increase the rate and level of activation of the cyclin-dependent kinases CDK2 and CDC2, and their respective active association with cyclins E and A or cyclin B. HBx is also shown to override or greatly reduce serum dependence for cell cycle activation. Both HBx and serum were found to require activation of RAS to stimulate cell cycling, but only HBx could shorten checkpoint intervals. HBx therefore stimulates cell proliferation by activating RAS and a second unknown effector, which may be related to its reported ability to induce prolonged activation of JUN or to interact with cellular p53 protein. These data suggest a molecular mechanism by which HBx likely contributes to viral carcinogenesis. By deregulating checkpoint controls, HBx could participate in the selection of cells that are genetically unstable, some of which would accumulate unrepaired transforming mutations.