A Conserved DNA Damage Response Pathway Responsible for Coupling the Cell Division Cycle to the Circadian and Metabolic Cycles

The circadian clock drives endogenous oscillations of cellular and physiological processes with a periodicity of approximately 24 h. Progression of the cell division cycle (CDC) has been found to be coupled to the circadian clock, and it has been postulated that gating of the CDC by the circadian cycle may have evolved to protect DNA from the mutagenic effects of ultraviolet light. When grown under nutrient-limiting conditions in a chemostat, prototrophic strains of budding yeast, Saccharomyces cerevisiae, adopt a robust metabolic cycle of ultradian dimensions that temporally compartmentalizes essential cellular events. The CDC is gated by this yeast metabolic cycle (YMC), with DNA replication strictly segregated away from the oxidative phase when cells are actively respiring. Mutants impaired in such gating allow DNA replication to take place during the respiratory phase of the YMC and have been found to suffer significantly elevated rates of spontaneous mutation. Analogous to the circadian cycle, the YMC also employs the conserved DNA checkpoint kinase Rad53/Chk2 to facilitate coupling with the CDC. These studies highlight an evolutionarily conserved mechanism that seems to confine cell division to particular temporal windows to prevent DNA damage. We hypothesize that DNA damage itself might constitute a “zeitgeber”, or time giver, for both the circadian cycle and the metabolic cycle. We discuss these findings in the context of a unifying theme underlying the circadian and metabolic cycles, and explore the relevance of cell cycle gating to human diseases including cancer.     

Coupling among growth rate response, metabolic cycle, and cell division cycle in yeast

We studied the steady-state responses to changes in growth rate of yeast when ethanol is the sole source of carbon and energy. Analysis of these data, together with data from studies where glucose was the carbon source, allowed us to distinguish a "universal" growth rate response (GRR) common to all media studied from a GRR specific to the carbon source. Genes with positive universal GRR include ribosomal, translation, and mitochondrial genes, and those with negative GRR include autophagy, vacuolar, and stress response genes. The carbon source-specific GRR genes control mitochondrial function, peroxisomes, and synthesis of vitamins and cofactors, suggesting this response may reflect the intensity of oxidative metabolism. All genes with universal GRR, which comprise 25% of the genome, are expressed periodically in the yeast metabolic cycle (YMC). We propose that the universal GRR may be accounted for by changes in the relative durations of the YMC phases. This idea is supported by oxygen consumption data from metabolically synchronized cultures with doubling times ranging from 5 to 14 h. We found that the high oxygen consumption phase of the YMC can coincide exactly with the S phase of the cell division cycle, suggesting that oxidative metabolism and DNA replication are not incompatible.     

Ability of human CDC25B phosphatase splice variants to replace the function of the fission yeast Cdc25 cell cycle regulator

CDC25 phosphatases are essential and evolutionary-conserved actors of the eukaryotic cell cycle control. To examine and compare the properties of three splicing variants of human CDC25B, recombinant fission yeast strains expressing the human proteins in place of the endogenous Cdc25 were generated and characterized. We report, that the three CDC25B variants: (i) efficiently replace the yeast counterpart in vegetative growth, (ii) partly restore the gamma and UV radiation DNA damage-activated checkpoint, (iii) fail to restore the DNA replication checkpoint activated by hydroxyurea. Although these yeast strains do not reveal the specific functions of the human CDC25B variants, they should provide useful screening tools for the identification of new cell cycle regulators and pharmacological inhibitors of CDC25 phosphatase.     

Controls over the timing of DNA replication during the cell cycle of fission yeast

The controls acting over the timing of DNA replication (S) during the cell cycle have been investigated in the fission yeast Schizosaccharomyces pombe. The cell size at which DNA replication takes place has been determined in a number of experimental situations such as growth of nitrogen-starved cells, spore germination and synchronous culture of wee mutant and wild-type strains. It is shown that in wee mutant strains and in wild type grown under conditions in which the cells are small, DNA replication takes place in cells of the same size. This suggests that there is a minimum cell size beneath which the cell cannot initiate DNA replication and it is this control which determines the timing of S during the cell cycle of the wee mutant. Fast growing wild-type cells are too large for this size control to be expressed. In these cells the timing of S may be controlled by the completion of the previous nuclear division coupled with a requirement for a minimum period in G1. Thus in S. pombe there are two different controls over the timing of S, either of which can be operative depending upon the size of the cell at cell division. It is suggested that these two controls may form a useful conceptual framework for considering the timing control over S in mammalian cells.     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae.

Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.     

A survey of essential gene function in the yeast cell division cycle

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO(7) promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication.     

A cell cycle checkpoint monitors cell morphogenesis in budding yeast

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.     

细胞周期研究的新进展

细胞周期研究的新进展陆长德（中国科学院上海生物化学研究所２０００３１）主要来自三方面的研究以及它们之间的相互交叉对于细胞周期研究的进展起了很大的作用。十多年来酵母分子遗传学的研究鉴定了许多与细胞周期的控制有关的基因,提供了许多突变株（如ＣＤＣ）;１９８８年对蛙卵成熟促进因子ＭＰＦ成分的鉴定和对它生物学功能的确定使人们对细胞周期的认识有了一个飞跃;人类的致癌基因（如Ｔａｇ）,肿瘤抑制基因（如p53,pRB)以及其他一些疾病(如对电离辐射敏感的遗传病,AT的分子机制的研究也大大地促进了细胞周期的研究。     

Cell cycle parameters of Proteus mirabilis: interdependence of the biosynthetic cell cycle and the interdivision cycle.

We investigated the time periods of DNA replication, lateral cell wall extension, and septum formation within the cell cycle of Proteus mirabilis. Cells were cultivated under three different conditions, yielding interdivision times of approximately 55, 57, and 160 min, respectively. Synchrony was achieved by sucrose density gradient centrifugation. The time periods were estimated by division inhibition studies with cephalexin, mecillinam, and nalidixic acid. In addition, DNA replication was measured by thymidine incorporation, and murein biosynthesis was measured by incorporation of N-acetylglucosamine into sodium dodecyl sulfate-insoluble murein sacculi. At interdivision times of 55 to 57 min murein biosynthesis for reproduction of a unit cell lasted longer than the interdivision time itself, whereas DNA replication finished within 40 min. Surprisingly, inhibition of DNA replication by nalidixic acid did not inhibit the subsequent cell division but rather the one after that. Because P. mirabilis fails to express several reactions of the recA-dependent SOS functions known from Escherichia coli, the drug allowed us to determine which DNA replication period actually governed which cell division. Taken together, the results indicate that at an interdivision time of 55 to 57 min, the biosynthetic cell cycle of P. mirabilis lasts approximately 120 min. To achieve the observed interdivision time, it is necessary that two subsequent biosynthetic cell cycles be tightly interlocked. The implications of these findings for the regulation of the cell cycle are discussed.     

Cell cycle characteristics of thermophilic archaea.

We have performed a cell cycle analysis of organisms from the Archaea domain. Exponentially growing cells of the thermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius were analyzed by flow cytometry, and several unusual cell cycle characteristics were found. The cells initiated chromosome replication shortly after cell division such that the proportion of cells with a single chromosome equivalent was low in the population. The postreplication period was found to be long; i.e., there was a considerable time interval from termination of chromosome replication until cell division. A further unusual feature was that cells in stationary phase contained two genome equivalents, showing that they entered the resting stage during the postreplication period. Also, a reduction in cellular light scatter was observed during entry into stationary phase, which appeared to reflect changes not only in cell size but also in morphology and/or composition. Finally, the in vivo organization of the chromosome DNA appeared to be different from that of eubacteria, as revealed by variation in the relative binding efficiency of different DNA stains.     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.     

A dependent pathway of gene functions leading to chromosome segregation in Saccharomyces cerevisiae

Methyl-benzimidazole-2-ylcarbamate (MBC) inhibits the mitotic cell cycle of Saccharomyces cerevisiae at a stage subsequent to DNA synthesis and before the completion of nuclear division (Quinlan, R. A., C. I. Pogson, and K, Gull, 1980, J Cell Sci., 46: 341-352). The step in the cell cycle that is sensitive to MBC inhibition was ordered to reciprocal shift experiments with respect to the step catalyzed by cdc gene products. Execution of the CDC7 step is required for the initiation of DNA synthesis and for completion of the MBC-sensitive step. Results obtained with mutants (cdc2, 6, 8, 9, and 21) defective in DNA replication and with an inhibitor of DNA replication (hydroxyurea) suggest that some DNA replication required for execution of the MBC-sensitive step but that the completion of replication is not. Of particular interest were mutants (cdc5, 13, 14, 15, 16, 17, and 23) that arrest cell division after DNA replication but before nuclear division since previous experiments had not been able to resolve the pathway of events in this part of the cell cycle. Execution of the CDC17 step was found to be a prerequisite for execution of the MBC- sensitive step; the CDC13, 16 and 23 steps are executed independently of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the CDC14 and 23 steps. These results considerably extend the dependent pathway of events that constitute the cell cycle of S. cerevisiae.     

Colcemid-resistant mutants of fission yeast have an altered cell cycle

Cell-cycle progression is altered in some colcemid-resistant mutants of fission yeast. The duration of particular stages of the cell cycle is different but total doubling time is unchanged from that of wild type. Cell-plate formation is prolonged relative to wild type but concomitant DNA synthesis is not affected. Separation of daughter cells is inhibited in some strains, giving rise to unusual cell morphologies. Control of cell division is altered in two ways: Critical size for division is increased. The probability of division a function of size is decreased.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

Cell cycle characteristics of crenarchaeota: unity among diversity

The hyperthermophilic archaea Acidianus hospitalis, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrobaculum calidifontis, and Sulfolobus tokodaii representing three different orders in the phylum Crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. The overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generation time. Thus, in all Crenarchaeota analyzed to date, cell division and initiation of chromosome replication occur in close succession, and a long time interval separates termination of replication from cell division. In Pyrobaculum, chromosome segregation overlapped with or closely followed DNA replication, and further genome separation appeared to occur concomitant with cellular growth. Cell division in P. aerophilum took place without visible constriction.     

Chromosome replication during the division cycle in slowly growing, steady-state cultures of three Escherichia coli B/r strains.

The period of DNA synthesis C during the cell cycle was determined over a broad range of generation times in slowly growing, steady-state batch cultures in the exponential phase and in chemostat cultures of three strains of Escherichia coli, strains B/r A, B/r K, and B/r TT, utilizing measurements of average amounts of DNA per cell and cell survival after radioactive decay of 125I incorporated into the DNA of synthesizing cells. At each growth rate, values for cell survival and for C periods were the same within experimental errors for the three strains. The length of the DNA synthesis period increased linearly with generation (doubling) time T of the culture and approached a limiting value of C = 0.36T at very long generation times. In very slowly growing cultures, DNA replication was limited almost entirely to the final third of the cell cycle. D periods, between termination of DNA replication and cell division, were found to be relatively short at all growth rates for each strain. Average amounts of DNA per cell measured in slowly growing cultures of strains B/r A and B/r TT were indistinguishable from results for strain B/r K at the same growth rates. Amounts of DNA per cell calculated from the cell survival values alone are completely consistent with the measured DNA per cell.