Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia–telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase ɛ and is required for chromosomal replication but not for the S phase checkpoint

In fission yeast both DNA polymerase alpha (pol α) and delta (pol δ) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol ɛ) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol ɛ (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29–39]. We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol ɛ does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol α rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol α, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.

Cdc25 Inhibited In Vivo and In Vitro by Checkpoint Kinases Cds1 and Chk1

In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S–M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S–M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G2–M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S–M replication and G2–M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.

Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

Functional Domains of Rep2, a Transcriptional Activator Subunit for Res2–Cdc10, Controlling the Cell Cycle “Start”

In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2+ gene, isolated as a multicopy suppressor of a temperature-sensitive cdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2–Cdc10 complex. To identify the rep2+ function and molecularly define its domain organization, we reconstituted the Res2–Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2–Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2 deletion mutant of fission yeast.

The Human G2 Checkpoint Control Protein hRAD9 Is a Nuclear Phosphoprotein That Forms Complexes with hRAD1 and hHUS1

Eukaryotic cells actively block entry into mitosis in the presence of DNA damage or incompletely replicated DNA. This response is mediated by signal transduction cascades called cell cycle checkpoints. We show here that the human checkpoint control protein hRAD9 physically associates with two other checkpoint control proteins, hRAD1 and hHUS1. Furthermore, hRAD1 and hHUS1 themselves interact, analogously to their fission yeast homologues Rad1 and Hus1. We also show that hRAD9 is present in multiple phosphorylation forms in vivo. These phosphorylated forms are present in tissue culture cells that have not been exposed to exogenous sources of DNA damage, but it remains possible that endogenous damage or naturally occurring replication intermediates cause the observed phosphorylation. Finally, we show that hRAD9 is a nuclear protein, indicating that in this signal transduction pathway, hRAD9 is physically proximal to the upstream (DNA damage) signal rather than to the downstream, cytoplasmic, cell cycle machinery.

Isolated Mammalian and Schizosaccharomyces pombe Ran-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein–mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1− yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1− yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

Plo1 Kinase Recruitment to the Spindle Pole Body and Its Role in Cell Division in Schizosaccharomyces pombe

Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis (Ohkura et al., 1995; Bahler et al., 1998). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 (Hudson et al., 1990). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression of plo1+ activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation.

Cell Cycle–regulated Proteolysis of Mitotic Target Proteins

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase–anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C–dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1–S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

A WD Repeat Protein Controls the Cell Cycle and Differentiation by Negatively Regulating Cdc2/B-Type Cyclin Complexes

In the fission yeast Schizosaccharomyces pombe, p34cdc2 plays a central role controlling the cell cycle. We recently isolated a new gene named srw1+, capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34cdc2. Cells lacking srw1+ are sterile and defective in cell cycle controls. When starved for nitrogen source, they fail to effectively arrest in G1 and die of accelerated mitotic catastrophe if regulation of p34cdc2/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34cdc2. srw1+ shares functional similarity with rum1+, having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34cdc2 and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments.

Identification of a Second Myosin-II in Schizosaccharomyces pombe:: Myp2p Is Conditionally Required for Cytokinesis

As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring. We cloned myp2+, a myosin-II in Schizosaccharomyces pombe, conditionally required for cytokinesis. myp2+, the second myosin-II identified in S. pombe, does not completely overlap in function with myo2+. The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family. The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs. However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines. Disruption of myp2+ is not lethal but under nutrient limiting conditions cells lacking myp2+ function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis. The presence of salt enhances these morphological defects. Additionally, Δmyp2 cells are cold sensitive in high salt, failing to form colonies at 17°C. Thus, myp2+ is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis. Additionally, we constructed double mutants of Δmyp2 with temperature-sensitive mutant strains defective in cytokinesis. We observed synthetic lethal interactions between Δmyp2 and three alleles of cdc11ts, as well as more modest synthetic interactions with cdc14ts and cdc16ts, implicating myp2+ function for efficient cytokinesis under normal conditions.

Nuclear Localization of Schizosaccharomyces pombe Mcm2/Cdc19p Requires MCM Complex Assembly

The minichromosome maintenance (MCM) proteins MCM2–MCM7 are conserved eukaryotic replication factors that assemble in a heterohexameric complex. In fission yeast, these proteins are nuclear throughout the cell cycle. In studying the mechanism that regulates assembly of the MCM complex, we analyzed the cis and trans elements required for nuclear localization of a single subunit, Mcm2p. Mutation of any single mcm gene leads to redistribution of wild-type MCM subunits to the cytoplasm, and this redistribution depends on an active nuclear export system. We identified the nuclear localization signal sequences of Mcm2p and showed that these are required for nuclear targeting of other MCM subunits. In turn, Mcm2p must associate with other MCM proteins for its proper localization; nuclear localization of MCM proteins thus requires assembly of MCM proteins in a complex. We suggest that coupling complex assembly to nuclear targeting and retention ensures that only intact heterohexameric MCM complexes remain nuclear.

Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro, ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.

Characterization of Schizosaccharomyces pombe RNA triphosphatase

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the β–γ phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast Schizosaccharomyces pombe. Pct1p hydrolyzes the γ phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and Pi in the presence of manganese or cobalt (Km = 19 µM ATP; kcat = 67 s–1). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I0.5 = 1 mM), pyrophosphate (I0.5 = 0.4 mM) and tripolyphosphate (I0.5 = 30 µM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.

The Schizosaccharomyces pombe spo20+ Gene Encoding a Homologue of Saccharomyces cerevisiae Sec14 Plays an Important Role in Forespore Membrane Formation

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+ is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20+ gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.