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.     

Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication

In eukaryotes, it is unknown whether mismatch repair (MMR) is temporally coupled to DNA replication and how strand-specific MMR is directed. We fused Saccharomyces cerevisiae MSH6 with cyclins to restrict the availability of the Msh2-Msh6 mismatch recognition complex to either S phase or G2/M phase of the cell cycle. The Msh6-S cyclin fusion was proficient for suppressing mutations at three loci that replicate at mid-S phase, whereas the Msh6-G2/M cyclin fusion was defective. However, the Msh6-G2/M cyclin fusion was functional for MMR at a very late-replicating region of the genome. In contrast, the heteroduplex rejection function of MMR during recombination was partially functional during both S phase and G2/M phase. These results indicate a temporal coupling of MMR, but not heteroduplex rejection, to DNA replication.     

DNA replication-independent silencing in S. cerevisiae

In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G(2)/M phase, which has been widely assumed to be DNA replication. To determine whether DNA replication through a silent mating-type locus, HMRa, is required for silencing to be established, we monitored heterochromatin formation at HMRa on a chromosome and on a nonreplicating extrachromosomal cassette as cells passed through S phase. Cells that passed through S phase established silencing at both the chromosomal HMRa locus and the extrachromosomal HMRa locus with equal efficiency. Thus, in contrast to the prevailing view, the establishment of silencing occurred in the absence of passage of the DNA replication fork through or near the HMR locus, but retained a cell cycle dependence.     

Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells

Caffeine was shown to induce mitotic events in mammalian cells before DNA replication (S phase) was completed. Synchronized BHK cells that were arrested in early S phase underwent premature chromosome condensation, nuclear envelope breakdown, morphological "rounding up," and mitosis-specific phosphoprotein synthesis when they were exposed to caffeine. These mitotic responses occurred only after the cells had entered S phase and only while DNA synthesis was inhibited by more than 70 percent. Inhibitors of protein synthesis blocked these caffeine-induced events, while inhibitors of RNA synthesis had little effect. These results suggest that caffeine induces the translation or stabilizes the protein product (or products) of mitosis-related RNA that accumulates in S-phase cells when DNA replication is suppressed. The ability to chemically manipulate the onset of mitosis should be useful for studying the regulation of this event in mammalian cells.     

Self-assembling protein microarrays

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.     

S phase of the cell cycle

In each cell cycle the complex structure of the chromosome must be replicated accurately. In the last few years there have been major advances in understanding eukaryotic chromosome replication. Patterns of replication origins have been mapped accurately in yeast chromosomes. Cellular replication proteins have been identified by fractionating cell extracts that replicate viral DNA templates in vitro. Cell-free systems that initiate eukaryotic DNA replication in vitro have demonstrated the importance of complex nuclear architecture in the control of DNA replication. Although the events of S phase were relatively neglected for many years, knowledge of DNA replication is now advancing rapidly in step with other phases of the cell cycle.     

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.     

Inhibition of eukaryotic DNA replication by geminin binding to Cdt1

In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.     

Pol kappa: A DNA polymerase required for sister chromatid cohesion

Establishment of cohesion between sister chromatids is coupled to replication fork passage through an unknown mechanism. Here we report that TRF4, an evolutionarily conserved gene necessary for chromosome segregation, encodes a DNA polymerase with beta-polymerase-like properties. A double mutant in the redundant homologs, TRF4 and TRF5, is unable to complete S phase, whereas a trf4 single mutant completes a presumably defective S phase that results in a failure of cohesion between the replicated sister chromatids. This suggests that TRFs are a key link in the coordination between DNA replication and sister chromatid cohesion. Trf4 and Trf5 represent the fourth class of essential nuclear DNA polymerases (designated DNA polymerase kappa) in Saccharomyces cerevisiae and probably in all eukaryotes.     

Activation of the DNA replication checkpoint through RNA synthesis by primase

When DNA replication is inhibited during the synthesis (S) phase of the cell cycle, a signaling pathway (checkpoint) is activated that serves to prevent mitosis from initiating before completion of replication. This replication checkpoint acts by down-regulating the activity of the mitotic inducer cdc2-cyclin B. Here, we report the relation between chromatin structure and induction of the replication checkpoint. Chromatin was competent to initiate a checkpoint response only after the DNA was unwound and DNA polymerase alpha had been loaded. Checkpoint induction did not require new DNA synthesis on the unwound template strand but did require RNA primer synthesis by primase. These findings identify the RNA portion of the primer as an important component of the signal that activates the replication checkpoint.     

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.     

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.     

Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets

Damage to the vessel wall is a signal for endothelial migration and replication and for platelet release at the site of injury. Addition of transforming growth factor-beta (TGF-beta) purified from platelets to growing aortic endothelial cells inhibited [3H]thymidine incorporation in a concentration-dependent manner. A transient inhibition of DNA synthesis was also observed in response to wounding; cell migration and replication are inhibited during the first 24 hours after wounding. By 48 hours after wounding both TGF-beta-treated and -untreated cultures showed similar responses. Flow microfluorimetric analysis of cell cycle distribution indicated that after 24 hours of exposure to TGF-beta the cells were blocked from entering S phase, and the fraction of cells in G1 was increased. The inhibition of the initiation of regeneration by TGF-beta could allow time for recruitment of smooth muscle cells into the site of injury by other platelet components.     

Temporal coordination of DNA replication with enzyme synthesis in diploid and heteroploid cells

The rate of DNA synthesis in the S phase of growth of synchronized diploid Chinese hamster cells shows two maximums, while in heteroploid hamster cells the DNA replication rate is constant. In diploid cells a reciprocal relationship exists between maximum DNA synthetic rates and maximum lactate dyhydrogenase and thymidine kinase enzyme levels. Enzyme activity in heteroploid cells increases continuously through the cell cycle with no evidence of oscillations. It seems possible that these differences in molecular organization may accompany or precede the transition to heteroploidy.     

Rapid destruction of human Cdc25A in response to DNA damage

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.     

广西不同生境蔗田蔗茎红粉蚧的种群动态

【目的】分析蔗茎红粉蚧在广西蔗区不同生境蔗田的种群动态,了解其在蔗田的发生规律,为生产上监测和控制该粉蚧提供参考。【方法】在广西上思县蔗区于2012年7-12月调查新台糖22号宿根1年蔗蔗茎红粉蚧的虫口密度和为害株率,2013年7～12月调查新台糖22号宿根1年蔗和粤糖60号新植蔗蔗茎红粉蚧的虫口密度;在扶绥县蔗区于2013年7～12月调查新台糖22号新植蔗与宿根1年蔗蔗茎红粉蚧的虫口密度。虫口密度调查为在蔗田设8个取样点,每点相隔3行,每取样点选取相近3株甘蔗,调查和记录蔗茎红粉蚧若虫和成虫的发生数量;为害株率为顺序调查同行100株甘蔗中发生蔗茎红粉蚧的株数,重复4次。每半个月调查一次,直至甘蔗收获。【结果】上思县蔗区调查结果,2012年蔗茎红粉蚧于7～8月发生数量较多,最高为100．50头/株,在9月下旬后发生数量明显减少;为害株率为24．75％78．50％,8-11月高达60％以上;2013年蔗茎红粉蚧在7～8月发生较多,新植蔗和宿根蔗的最大虫口密度分别为44．51和27．96头／株。扶绥县蔗区2013年调查结果,蔗茎红粉蚧发生数量也以7～8月较多,该粉蚧在宿根蔗上的最大虫口密度显著高于新植蔗,分别为125．87和25．08头／株。【结论】每年7～8月是广西蔗区蔗茎红粉蚧自然种群增长最快的时期,生产上应密切关注这一时期的蔗茎红粉蚧种群动态,并根据其危害情况及时采取必要的防治措施。     

Growth of prochlorococcus, a photosynthetic prokaryote, in the equatorial pacific ocean

The cell cycle of Prochlorococcus, a prokaryote that accounts for a sizable fraction of the photosynthetic biomass in the eastern equatorial Pacific, progressed in phase with the daily light cycle. DNA replication occurred in the afternoon and cell division occurred at night. Growth rates were maximal (about one doubling per day) at 30 meters and decreased toward the surface and the bottom of the ocean. Estimated Prochlorococcus production varied between 174 and 498 milligrams of carbon per square meter per day and accounted for 5 to 19 percent of total gross primary production at the equator. Because Prochlorococcus multiplies close to its maximum possible rate, it is probably not severely nutrient-limited in this region of the oceans.     

麝香草酚对酿酒酵母表达谱影响的研究

麝香草酚是从百里香和陈皮中的提取的精油,具有抗病原真菌的活性。本实验应用基因芯片技术从全基因组水平考察了麝香草酚对酿酒酵母表达谱的影响,并用聚类软件T-profiler,分析了基因芯片数据。结果表明,酵母细胞中与磷脂合成、铁吸收等有关的基因的表达受到麝香草酚的影响。     

Nonhistone chromosomal protein synthesis: utilization of preexisting and newly transcribed messenger RNA's

Treatment of HeLa S(3) cells with actinomycin D during mitosis suppresses the synthesis of several major classes of nonhistone chromosomal proteins during the subsequent period before DNA replication, but allows the synthesis of other species of these proteins. Such results are consistent with the utilization of preexisting, as well as newly transcribed, messenger RNA's for nonhistone chromosomal protein synthesis during the prereplicative phase of the cell cycle.