Est1p as a cell cycle-regulated activator of telomere-bound telomerase

In Saccharomyces cerevisiae, the telomerase components Est2p, TLC1 RNA, Est1p, and Est3p are thought to form a complex that acts late during chromosome replication (S phase) upon recruitment by Cdc13p, a telomeric DNA binding protein. Consistent with this model, we show that Est1p, Est2p, and Cdc13p are telomere-associated at this time. However, Est2p, but not Est1p, also binds telomeres before late S phase. The cdc13-2 allele has been proposed to be defective in recruitment, yet Est1p and Est2p telomere association persists in cdc13-2 cells. These findings suggest a model in which Est1p binds telomeres late in S phase and interacts with Cdc13p to convert inactive, telomere-bound Est2p to an active form.     

Oscillatory dynamics of Cdc42 GTPase in the control of polarized growth

Cells promote polarized growth by activation of Rho-family protein Cdc42 at the cell membrane. We combined experiments and modeling to study bipolar growth initiation in fission yeast. Concentrations of a fluorescent marker for active Cdc42, Cdc42 protein, Cdc42-activator Scd1, and scaffold protein Scd2 exhibited anticorrelated fluctuations and oscillations with a 5-minute average period at polarized cell tips. These dynamics indicate competition for active Cdc42 or its regulators and the presence of positive and delayed negative feedbacks. Cdc42 oscillations and spatial distribution were sensitive to the amounts of Cdc42-activator Gef1 and to the activity of Cdc42-dependent kinase Pak1, a negative regulator. Feedbacks regulating Cdc42 oscillations and spatial self-organization appear to provide a flexible mechanism for fission yeast cells to explore polarization states and to control their morphology.     

Requirement of the prolyl isomerase Pin1 for the replication checkpoint

The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.     

The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae

Cell division is arrested in many organisms in response to DNA damage. Examinations of the genetic basis for this response in the yeast Saccharomyces cerevisiae indicate that the RAD9 gene product is essential for arrest of cell division induced by DNA damage. Wild-type haploid cells irradiated with x-rays either arrest or delay cell division in the G2 phase of the cell cycle. Irradiated G1 and M phase haploid cells arrest irreversibly in G2 and die, whereas irradiated G2 phase haploid cells delay in G2 for a time proportional to the extent of damage before resuming cell division. In contrast, irradiated rad9 cells in any phase of the cycle do not delay cell division in G2, but continue to divide for several generations and die. However, efficient DNA repair can occur in irradiated rad9 cells if irradiated cells are blocked for several hours in G2 by treatment with a microtubule poison. The RAD9-dependent response detects potentially lethal DNA damage and causes arrest of cells in G2 until such damage is repaired.     

Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation

Meiosis is a specialized cell division in which two chromosome segregation phases follow a single DNA replication phase. The budding yeast Polo-like kinase Cdc5 was found to be instrumental in establishing the meiosis I chromosome segregation program. Cdc5 was required to phosphorylate and remove meiotic cohesin from chromosomes. Furthermore, in the absence of CDC5 kinetochores were bioriented during meiosis I, and Mam1, a protein essential for coorientation, failed to associate with kinetochores. Thus, sister-kinetochore coorientation and chromosome segregation during meiosis I are coupled through their dependence on CDC5.     

Protein tyrosine kinase Wee1B is essential for metaphase II exit in mouse oocytes

Waves of cyclin synthesis and degradation regulate the activity of Cdc2 protein kinase during the cell cycle. Cdc2 inactivation by Wee1B-mediated phosphorylation is necessary for arrest of the oocyte at G2-prophase, but it is unclear whether this regulation functions later during the metaphase-to-anaphase transition. We show that reactivation of a Wee1B pathway triggers the decrease in Cdc2 activity during egg activation. When Wee1B is down-regulated, oocytes fail to form a pronucleus in response to Ca(2+) signals. Calcium-calmodulin-dependent kinase II (CaMKII) activates Wee1B, and CaMKII-driven exit from metaphase II is inhibited by Wee1B down-regulation, demonstrating that exit from metaphase requires not only a proteolytic degradation of cyclin B but also the inhibitory phosphorylation of Cdc2 by Wee1B.     

Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56

Posttranslational modifications of the histone octamer play important roles in regulating responses to DNA damage. Here, we reveal that Saccharomyces cerevisiae Rtt109p promotes genome stability and resistance to DNA-damaging agents, and that it does this by functionally cooperating with the histone chaperone Asf1p to maintain normal chromatin structure. Furthermore, we show that, as for Asf1p, Rtt109p is required for histone H3 acetylation on lysine 56 (K56) in vivo. Moreover, we show that Rtt109p directly catalyzes this modification in vitro in a manner that is stimulated by Asf1p. These data establish Rtt109p as a member of a new class of histone acetyltransferases and show that its actions are critical for cell survival in the presence of DNA damage during S phase.     

Identification of the Cdc48•20S proteasome as an ancient AAA+ proteolytic machine

Proteasomes are the major energy-dependent proteolytic machines in the eukaryotic and archaeal domains of life. To execute protein degradation, the 20S core peptidase combines with the AAA+ ring of the 19S regulatory particle in eukarya or with the AAA+ proteasome-activating nucleotidase ring in some archaea. Here, we find that Cdc48 and 20S from the archaeon Thermoplasma acidophilum interact to form a functional proteasome. Cdc48 is an abundant and essential double-ring AAA+ molecular machine ubiquitously present in archaea, where its function has been uncertain, and in eukarya where Cdc48 participates by largely unknown mechanisms in diverse cellular processes, including multiple proteolytic pathways. Thus, proteolysis in collaboration with the 20S peptidase may represent an ancestral function of the Cdc48 family.     

ATM engages autodegradation of the E3 ubiquitin ligase COP1 after DNA damage

The ataxia telangiectasia mutated (ATM) protein kinase is a critical component of a DNA-damage response network configured to maintain genomic integrity. The abundance of an essential downstream effecter of this pathway, the tumor suppressor protein p53, is tightly regulated by controlled degradation through COP1 and other E3 ubiquitin ligases, such as MDM2 and Pirh2; however, the signal transduction pathway that regulates the COP1-p53 axis following DNA damage remains enigmatic. We observed that in response to DNA damage, ATM phosphorylated COP1 on Ser(387) and stimulated a rapid autodegradation mechanism. Ionizing radiation triggered an ATM-dependent movement of COP1 from the nucleus to the cytoplasm, and ATM-dependent phosphorylation of COP1 on Ser(387) was both necessary and sufficient to disrupt the COP1-p53 complex and subsequently to abrogate the ubiquitination and degradation of p53. Furthermore, phosphorylation of COP1 on Ser(387) was required to permit p53 to become stabilized and to exert its tumor suppressor properties in response to DNA damage.     

Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase

The regulation of DNA replication during the eukaryotic cell cycle was studied in a system where cell free replication of simian virus 40 (SV40) DNA was used as a model for chromosome replication. A factor, RF-S, was partially purified from human S phase cells based on its ability to activate DNA replication in extracts from G1 cells. RF-S contained a human homologue of the Schizosaccharomyces pombe p34cdc2 kinase, and this kinase was necessary for RF-S activity. The limiting step in activation of the p34 kinase at the G1 to S transition may be its association with a cyclin since addition of cyclin A to a G1 extract was sufficient to start DNA replication. These observations suggest that the role of p34cdc2 in controlling the start of DNA synthesis has been conserved in evolution.     

Postreplicative formation of cohesion is required for repair and induced by a single DNA break

Sister-chromatid cohesion, established during replication by the protein complex cohesin, is essential for both chromosome segregation and double-strand break (DSB) repair. Normally, cohesion formation is strictly limited to the S phase of the cell cycle, but DSBs can trigger cohesion also after DNA replication has been completed. The function of this damage-induced cohesion remains unknown. In this investigation, we show that damage-induced cohesion is essential for repair in postreplicative cells in yeast. Furthermore, it is established genome-wide after induction of a single DSB, and it is controlled by the DNA damage response and cohesin-regulating factors. We thus define a cohesion establishment pathway that is independent of DNA duplication and acts together with cohesion formed during replication in sister chromatid-based DSB repair.     

An origin unwinding activity regulates initiation of DNA replication during mammalian cell cycle

An in vitro assay was developed to study the positive factors that regulate the onset of DNA replication during the mammalian cell cycle. Extracts prepared from cells at defined positions in the cell cycle were used to examine the replication of SV40 DNA in a cell free system. Extracts prepared from S phase cells were ten times more efficient at initiating replication at the SV40 origin than were extracts from G1 cells, whereas elongation rates were similar in G1 and S reactions. At a discrete point in the cell cycle, just before the cell's entry into S, an activity appeared that was required, in conjunction with SV40 T antigen, for site specific initiation at the SV40 origin. This factor had a role in unwinding DNA at the replication origin.     

Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts

Heterokaryon studies suggest that senescent and quiescent human diploid fibroblasts (HDF) contain a common inhibitor of entry into S phase. DNA synthesis can be induced in senescent and quiescent HDF by fusing them with cells containing DNA viral oncogenes such as SV40 T antigen, adenovirus E1A, or human papillomavirus E7. Both senescent and quiescent HDF contained the unphosphorylated form (p110Rb) of the retinoblastoma protein, a putative inhibitor of proliferation. After serum stimulation, senescent HDF did not phosphorylate p110Rb and did not enter S phase, whereas quiescent HDF phosphorylated p110Rb and entered S phase. These findings, combined with the observations that T antigen, E1A, and E7 form complexes with, and presumably inactivate, unphosphorylated p110Rb, suggest that failure to phosphorylate p110Rb may be an immediate cause of failure to enter S phase in senescent HDF.     

原花青素影响Aβ25-35诱导PC12细胞周期变化与细胞凋亡的可能机制

目的探讨原花青素（Proanthocyanidins,PAC）对β-淀粉样肽（25—35）[β amyloid peptide-（25—35）,Aβ25-35]诱导体外血清饥饿培养的PCI2细胞周期异常与凋亡保护作用的可能机制。方法种人培养瓶或板的PC12细胞贴壁后用常用血清饥饿培养使细胞同步于岛期,30mg／L的PAC预处理血清饥饿培养的PC12细胞,加入终浓度为25μmol／L Aβ25-35处理0～20h,通过RT-PCR和Western blot从mRNA及蛋白水平检测细胞周期蛋白依赖性激酶4（Cyclin—dependent kinase-4,CDK4）、磷酸化的视网膜纤维母细胞瘤蛋白（phosphorylated Retinoblastoma protein,pRb）、腺病毒E2启动子结合因子1（Adenovirus E2 factor-1,E2 F1）、B细胞淋巴瘤／白血病关联X蛋白（B-cell lymphoma／leukemia-2 Associated X protein,bax）基因表达的变化。结果与Aβ25—35诱导组比较,CDK4、E2F1、bax mRNA表达和CDK4、pR6、Bax蛋白表达降低。结论PAC可能通过下调CDK4、pRb、E2F1的表达,从而降低bax的活化,对Aβ25-35诱导的PC12细胞周期异常与凋亡起保护作用的。     

G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.     

α-三联噻吩对斜纹夜蛾SL细胞线粒体膜电位及细胞周期的影响#br#

【目的】研究α-三联噻吩（α-T）对斜纹夜蛾（Spodoptera litura）SL细胞线粒体膜电位及细胞周期的影响。【方法】通过Rhodamine123染色和流式细胞术（flow cytometry,FCM）研究α-T处理SL细胞24 h和48 h后细胞线粒体膜电位的变化,并通过PI染色和FCM,测定α-T处理SL细胞24 h和48 h后细胞周期各时相百分率的变化。【结果】光活化后的α-T处理SL细胞24 h后,0.0625 μg•mL-1浓度和0.1250 μg•mL-1浓度处理组细胞线粒体膜电位的变化幅度不大,而0.5000 μg•mL-1浓度处理组细胞线粒体膜电位明显升高,48 h后高浓度处理组（1.0000 μg•mL-1）导致线粒体膜电位明显去极化;处理24 h后,低浓度（0.0625 μg•mL-1）处理组细胞被阻滞于S期,而高于此浓度的光照处理组细胞被阻滞于G2/M期,48 h后,浓度≤0.1250 μg•mL-1的光照处理细胞被阻滞于G2/M期,而浓度＞0.1250 μg•mL-1的光照处理组细胞被阻滞于S期。【结论】SL细胞经α-T处理后,细胞处于一系列复杂的动态变化中。当细胞本身不能扭转ROS造成的氧化损伤时,细胞线粒体膜电位和细胞周期时相即出现较大幅度的变化,这种变化决定着细胞的受损程度和细胞的死亡途径。     

Activation of endogenous Cdc42 visualized in living cells

Signaling proteins are tightly regulated spatially and temporally to perform multiple functions. For Cdc42 and other guanosine triphosphatases, the subcellular location of activation is a critical determinant of cell behavior. However, current approaches are limited in their ability to examine the dynamics of Cdc42 activity in living cells. We report the development of a biosensor capable of visualizing the changing activation of endogenous, unlabeled Cdc42 in living cells. With the use of a dye that reports protein interactions, the biosensor revealed localized activation in the trans-Golgi apparatus, microtubule-dependent Cdc42 activation at the cell periphery, and activation kinetics precisely coordinated with cell extension and retraction.     

G1 events and regulation of cell proliferation

Cells prepare for S phase during the G1 phase of the cell cycle. Cell biological methods have provided knowledge of cycle kinetics and of substages of G1 that are determined by extracellular signals. Through the use of biochemical and molecular biological techniques to study effects of growth factors, oncogenes, and inhibitors, intracellular events during G1 that lead to DNA synthesis are rapidly being discovered. Many cells in vivo are in a quiescent state (G0), with unduplicated DNA. Cells can be activated to reenter the cycle during G1. Similarly, cells in culture can be shifted between G0 and G1. These switches in and out of G1 are the main determinants of post-embryonic cell proliferation rate and are defectively controlled in cancer cells.