Interfacial spin trapping in model membrane systems

The nature of mitochondrial DNA replication during the synchronous cell cycle in the yeast, $\text{Saccharomyces}\text{cerevisiae}$ has been investigated by examining the rate of labeled DNA precursor incorporation into specific segments of the mitochondrial genome at defined points during synchronous growth. The movement of label uptake from one area of the DNA to another at different times during the synchronous cell cycle indicates mitochondrial DNA replication to be a synchronous process during this time with most or all molecules at the same point in replication at any given time during the cell cycle.     

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis.

To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.     

Cdt1 Interactions in the Licensing Process: A Model for Dynamic Spatio-temporal Control of Licensing

Within each cell cycle, a cell must ensure that the processes of selection of replication origins (licensing) and initiation of DNA replication are well coordinated to prevent re-initiation of DNA replication from the same DNA segment during the same cell cycle. This is achieved by restricting the licensing process to G1 phase when the prereplicative complexes (preRCs) are assembled onto the origin DNA, while DNA replication is initiated only during S phase when de novo preRC assembly is blocked. Cdt1 is an important member of the preRC complex and its tight regulation through ubiquitin-dependent proteolysis and binding to its inhibitor Geminin ensure that Cdt1 will only be present in G1 phase, preventing relicensing of replication origins. We have recently reported that Cdt1 associates with chromatin in a dynamic way and recruits its inhibitor Geminin onto chromatin in vivo. Here we discuss how these dynamic Cdt1-chromatin interactions and the local recruitment of Geminin onto origins of replication by Cdt1 may provide a tight control of the licensing process in time and in space.     

Budding yeast Rap1, but not telomeric DNA,is inhibitory for multiple stages of DNA replication in vitro

Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process.     

Regulation of histone synthesis and nucleosome assembly

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.     

A moving DNA replication factory in Caulobacter crescentus

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.     

Size variations and correlation of different cell cycle events in slow-growing Escherichia coli.

Cell lengths have been determined at which cycle events occur in the slow-growing Escherichia coli B/r substrains A, K, and F26. The radioautographic and electron microscope analyses allowed determination of the variations in length at birth, initiation and termination of DNA replication, and initiation of the constriction process and of cell separation. In all three substrains the standard deviation increased between cell birth and initiation of DNA replication. From there on, the standard deviation remained relatively constant until cell separation. These observations are consistent with the presence of a deterministic phase during the cell cycle in which the cell sizes at initation of DNA replication and at cell division are correlated.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

Polo-like kinase 1 (Plk1): an Unexpected Player in DNA Replication

Regulation of cell cycle progression is important for the maintenance of genome integrity, and Polo-like kinases (Plks) have been identified as key regulators of this process. It is well established that Polo-like kinase 1 (Plk1) plays critical roles in mitosis but little is known about its functions at other stages of the cell cycle. Here we summarize the functions of Plk1 during DNA replication, focusing on the molecular events related to Origin Recognition Complex (ORC), the complex that is essential for the initiation of DNA replication. Within the context of Plk1 phosphorylation of Orc2, we also emphasize regulation of Orc2 in different organisms. This review is intended to provide some insight into how Plk1 coordinates DNA replication in S phase with chromosome segregation in mitosis, and orchestrates the cell cycle as a whole.     

Regulation of replication fork speed: Mechanisms and impact on genomic stability

Replication of DNA is a fundamental biological process that ensures precise duplication of the genome and thus safeguards inheritance. Any errors occurring during this process must be repaired before the cell divides, by activating the DNA damage response (DDR) machinery that detects and corrects the DNA lesions. Consistent with its significance, DNA replication is under stringent control, both spatial and temporal. Defined regions of the genome are replicated at specific times during S phase and the speed of replication fork progression is adjusted to fully replicate DNA in pace with the cell cycle. Insults that impair DNA replication cause replication stress (RS), which can lead to genomic instability and, potentially, to cell transformation. In this perspective, we review the current concept of replication stress, including the recent findings on the effects of accelerated fork speed and their impact on genomic (in)stability. We discuss in detail the Fork Speed Regulatory Network (FSRN), an integrated molecular machinery that regulates the velocity of DNA replication forks. Finally, we explore the potential for targeting FSRN components as an avenue to treat cancer.     

Regulation of DNA synthesis in bacteria: Analysis of the Bates/Kleckner licensing/initiation-mass model for cell cycle control

Bates and Kleckner have recently proposed that bacterial cell division is a licensing agent for a subsequent initiation of DNA replication. They also propose that initiation mass for DNA replication is not constant. These two proposals do not take into account older data showing that initiation of DNA replication can occur prior to the division event. This critical analysis is derived from measurements of DNA replication during the division cycle in cells growing at different, and more rapid, growth rates. Furthermore, mutants impaired in division can initiate DNA synthesis. The data presented by Bates and Kleckner do not support the proposal that initiation mass is variable, and the proposed pattern of DNA replication during the division cycle of the K12 cells analysed is not consistent with prior data on the pattern of DNA replication during the division cycle.     

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.     

Regulation of DNA replication during the cell cycle: roles of Cdc7 kinase and coupling of replication, recombination, and repair in response to replication fork arrest

DNA replication is central to cell growth, development, and generation of tissues and organs. Recent advances in understanding replication machinery have revealed striking conservation of components involved in the processes of DNA replication, from yeasts to human. The conservation extends even to bacteria for some basic components of replication apparatus. Eukaryotic DNA replication is regulated at various stages to ensure strict regulation during cell cycle. We have identified a novel mammalian kinase, Cdc7-ASK (Activator of S phase Kinase), that plays a key role at the entry into S phase as a molecular switch for DNA replication. This kinase is specifically activated during S phase and triggers the firing of DNA replication by phosphorylating an essential DNA helicase component of the replication complex. Environmental stresses such as DNA damages or depletion of essential nutrients for DNA synthesis lead to unscheduled arrest of DNA replication forks. In bacteria, this leads to induction of altered modes of DNA replication, which may repair DNA damages, facilitate reassembly of replication machinery at the stalled replication fork, or do both. In eukaryotes, blocking replication forks usually induces both checkpoint responses, which prevent premature progression of cell cycle events before precise completion of the preceding cell cycle stage, and the recombinational repair system for the lesions. Possible common bases in recognition of stalled replication forks in bacteria and eukaryotes will be discussed. Furthermore, we will discuss the potential of replication and checkpoint proteins as targets of anticancer agents as well as possible novel technology for stem cell amplification through manipulation of DNA replication.     

Preventing re-replication of chromosomal DNA

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2-7 proteins to form a pre-replicative complex. Mcm2-7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.     

The coupling of epigenome replication with DNA replication

In multicellular organisms, each cell contains the same DNA sequence, but with different epigenetic information that determines the cell specificity. Semi-conservative DNA replication faithfully copies the parental nucleotide sequence into two DNA daughter strands during each cell cycle. At the same time, epigenetic marks such as DNA methylation and histone modifications are either precisely transmitted to the daughter cells or dynamically changed during S-phase. Recent studies indicate that in each cell cycle, many DNA replication related proteins are involved in not only genomic but also epigenomic replication. Histone modification proteins, chromatin remodeling proteins, histone variants, and RNAs participate in the epigenomic replication during S-phase. As a consequence, epigenome replication is closely linked with DNA replication during S-phase.     

In vivo accumulation of cyclin A and cellular replication factors in autonomous parvovirus minute virus of mice-associated replication bodies

Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases alpha and delta in these bodies suggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruitment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement in activating the conversion of virion single-stranded DNA to the duplex replicative form.     

生理条件下的S期检查点

细胞周期检查点在细胞遭遇DNA损伤因子的攻击或遇到营养缺乏等不利因素作用时,能够暂时阻止或减慢细胞周期的进程,是细胞在长期进化中发展起来的抵御DNA损伤的重要机制.不仅如此,最近的研究表明,在正常生理条件下,存在一种S期检查点,对DNA复制的速度进行调控.从分子水平而言,这种调控作用可能是通过一系列细胞周期调控蛋白如ATR、9-1-1复合体、Chk1、Cdc25A和CDK2等的作用来实现的.这种调节作用对细胞至关重要,它使DNA复制速度不致于过快,从而减少复制过程中发生错误的几率,维护基因组的稳定性.     

Estimating the Total Rate of DNA Replication Using Branching Processes

Increasing the knowledge of various cell cycle kinetic parameters, such as the length of the cell cycle and its different phases, is of considerable importance for several purposes including tumor diagnostics and treatment in clinical health care and a deepened understanding of tumor growth mechanisms. Of particular interest as a prognostic factor in different cancer forms is the S phase, during which DNA is replicated. In the present paper, we estimate the DNA replication rate and the S phase length from bromodeoxyuridine-DNA flow cytometry data. The mathematical analysis is based on a branching process model, paired with an assumed gamma distribution for the S phase duration, with which the DNA distribution of S phase cells can be expressed in terms of the DNA replication rate. Flow cytometry data typically contains rather large measurement variations, however, and we employ nonparametric deconvolution to estimate the underlying DNA distribution of S phase cells; an estimate of the DNA replication rate is then provided by this distribution and the mathematical model.     

Mouse L cell mitochondrial DNA molecules are selected randomly for replication throughout the cell cycle

The number of mitochondrial DNA molecules in a cell population doubles at the same rate as the cell generation time. This could occur by a random selection of molecules for replication or by a process that ensures the replication of each individual molecule in the cell. We have investigated the rate at which mouse L cell mitochondrial DNA molecules labeled with 3H-thymidine during one round of replication are reselected for a second round of replication. Mouse L cells were labeled with 3H-thymidine for 2 hr, chased for various periods of time and then labeled with 5-bromodeoxyuridine for 4 hr immediately before mitochondrial DNA isolation. A constant fraction of 3H-thymidine-labeled mitochondrial DNA incorporated 5-bromodeoxyuridine after chase intervals ranging from 1.5-22 hr. This result demonstrates that mitochondrial DNA molecules replicated in a short time interval are randomly selected for later rounds of replication, and that replication of mitochondrial DNA continues throughout the cell cycle in mouse L cells.