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1.
Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules.
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The replication timing of a pair of natural alleles was compared at two alpha-tubulin loci of the Physarum plasmodium. Taking advantage of the naturally synchronous cell cycle of nuclei within the syncytial plasmodium, we analyzed the replication schedule of specific DNA fragments to a resolution of 10-min intervals within a 3-h S phase. At this level of resolution, differences in replication timing between polymorphic alleles at the same locus can be detected in a heterozygote. Specifically, the 3' region of the altA1 allele completes replication at between 20 and 40 min of S phase. The same region of the altA2 allele completes replication at between 40 and 80 min of S phase. In contrast, both alleles at the altB locus replicate concurrently within the first 10 to 15 min of S phase. Previous studies showed that both altA and altB are expressed in the plasmodium, their message levels peaking at mitosis, just minutes before the onset of S phase. However, altB message is detected at substantially higher levels than altA message on Northern (RNA) blots. The temporal windows over which the altA alleles each replicate are very broad in comparison with the levels of mitotic synchrony and altB replication synchrony in a single plasmodium. The allele-specific replication schedule of the altA locus demonstrates that the temporal organization of replicons is not strictly conserved between homologous chromosomes. 相似文献
2.
Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown. To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10. From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci. In early S phase, several hundred foci appeared throughout the nucleus. In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions. In the late S and G phases, GFP-Mcm10 was localized to nucleoli. Although (2)the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases. Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30-60 min. These results suggest that human Mcm10 is temporarily recruited to the replication sites 30-60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex. 相似文献
3.
Brylawski BP Cohen SM Horne H Irani N Cordeiro-Stone M Kaufman DG 《Journal of cellular biochemistry》2004,92(4):755-769
DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed. 相似文献
4.
5.
In detached Vicia embryos, the incorporation of [3-H] thymidine into DNA starts at about 25 h after the beginning of imbibition and reaches maximum at about 33 h. The DNA synthesized during the first replication phase was extracted. Alkaline sucrose density-gradient analyses of the DNA indicated the occurrence of several short pieces of rapidly labeled DNA having sedimentation values of approx. 10 S and 14 S, after a pulse for 5 to 10 min. Prolonged labeling and chase incubation led to a shift of the shorter fragments to longer ones of 19 S and 22 S or more, thus indicating the nature of intermediates during DNA replication of these short fragments. 相似文献
6.
Leach TJ Chotkowski HL Wotring MG Dilwith RL Glaser RL 《Molecular and cellular biology》2000,20(17):6308-6316
Heterochromatin is characteristically the last portion of the genome to be replicated. In polytene cells, heterochromatic sequences are underreplicated because S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells of Drosophila and may be the discontinuous molecules that form between fully replicated euchromatic and underreplicated heterochromatic regions of the chromosome. In this report, we characterize the temporal pattern of heterochromatic DNA truncation during development of polytene cells. Underreplication occurred during the first polytene S phase, yet DNA truncation, which was found within heterochromatic sequences of all four Drosophila chromosomes, did not occur until the second polytene S phase. DNA truncation was correlated with underreplication, since increasing the replication of satellite sequences with the cycE(1672) mutation caused decreased production of truncated DNAs. Finally, truncation of heterochromatic DNAs was neither quantitatively nor qualitatively affected by modifiers of position effect variegation including the Y chromosome, Su(var)205(2), parental origin, or temperature. We propose that heterochromatic satellite sequences present a barrier to DNA replication and that replication forks that transiently stall at such barriers in late S phase of diploid cells are left unresolved in the shortened S phase of polytene cells. DNA truncation then occurs in the second polytene S phase, when new replication forks extend to the position of forks left unresolved in the first polytene S phase. 相似文献
7.
Guilbaud G Rappailles A Baker A Chen CL Arneodo A Goldar A d'Aubenton-Carafa Y Thermes C Audit B Hyrien O 《PLoS computational biology》2011,7(12):e1002322
Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics. 相似文献
8.
Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos. 总被引:16,自引:4,他引:12
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We have analysed the replication of the chromosomal ribosomal DNA (rDNA) cluster in Xenopus embryos before the midblastula transition. Two-dimensional gel analysis showed that replication forks are associated with the nuclear matrix, as in differentiated cells, and gave no evidence for single-stranded replication intermediates (RIs). Bubbles, simple forks and double Ys were found in each restriction fragment analysed, showing that replication initiates and terminates without detectable sequence specificity. Quantification of the results and mathematical analysis showed that the average rDNA replicon replicates in 7.5 min and is 9-12 kbp in length. This time is close to the total S phase duration, and this replicon size is close to the maximum length of DNA which can be replicated from a single origin within this short S phase. We therefore infer that (i) most rDNA origins must be synchronously activated soon in S phase and (ii) origins must be evenly spaced, in order that no stretch of chromosomal DNA is left unreplicated at the end of S phase. Since origins are not specific sequences, it is suggested that this spatially and temporally concerted pattern of initiation matches some periodic chromatin folding, which itself need not rely on DNA sequence. 相似文献
9.
Three autonomously replicating plasmids carrying human genomic DNA and a vector derived from Epstein-Barr virus were studied by density labelling to determine the number of times per cell cycle these plasmids replicate in human cells. Each of the plasmids replicated semi-conservatively once per cell cycle. The results suggest that these human autonomously replicating sequences undergo replication following the same controls as chromosomal DNA and represent a good model system for studying chromosomal replication. We also determined the time within the S phase of the cell cycle that three of the plasmids replicate. Centromeric alpha sequences, which normally replicate late in S phase when in their chromosomal context, were found to replicate earlier when they mediate replication on an extrachromosomal vector. Reproducible patterns of replication within S phase were found for the plasmids, suggesting that the mechanism specifying time of replication may be subject to experimental analysis with this system. 相似文献
10.
Replication timing of DNA sequences associated with human centromeres and telomeres. 总被引:13,自引:3,他引:10
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K G Ten Hagen D M Gilbert H F Willard S N Cohen 《Molecular and cellular biology》1990,10(12):6348-6355
The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences. 相似文献
11.
Yu. B. Yurov 《Chromosoma》1979,74(3):347-353
DNA fiber autoradiography was used to measure the rate of replication fork progression along replication units in human diploid cells. The rate in different replication units differs very significantly and lies within the range 0.1 to 1.2 m/min. However, no significant changes were found in the rate of fork movement along single replication units operating during long intervals of S phase. Moreover, the fork progression rate is constant in many replication units of human cells. 相似文献
12.
Transmission electron microscopic techniques were used to study the spatial distribution of replicons and the ultrastructure of chromatin in the S phase genome of cellular blastoderm Drosophila melanogaster embryos. We observed chromatin exhibiting distinct bifurcations along each fiber during the initial 20 min of the first cell cycle of blastulation. We interpreted the “bubble-like” configurations produced by adjacent bifurcations as intermediate structures in chromatin replication (that is, replicons). We observed homologous ribonucleoprotein (RNP) fiber arrays on both chromatid arms within some replicons, implying DNA sequence homology and reinforcing the identification of such arms as daughter chromatid fibers. We did not observe replicon configurations on chromatin obtained from embryos staged at more than 20 min into cellular blastulation. Daughter chromatid fibers, however, were identified by the presence of identical RNP fiber arrays on chromatid strands arranged in parallel on the electron microscope grid.We examined the distribution of replicon structures on the cellular blastoderm genome and compared it with electron microscopic data on DNA replication in cleavage embryos (Blumenthal, Kriegstein and Hogness, 1973). S phase is completed in slightly < 4 min during cleavage, or approximately one fifth the time required for DNA synthesis in cellular blastoderm embryos. The mean distance separating adjacent replication origins at cellularization was estimated to be 10.6 kilobases (kb), a value 35% greater than the 7.9 kb inter-origin average determined for cleavage embryos. In contrast to the near-simultaneous activation of replication origins during cleavage replication, we observed that replication origins are not activated synchronously at cellular blastulation. We concluded that the marked increase in the duration of S phase is effected by a reduction in the frequency of replication activation events which occur asynchronously during genome replication at cellularization.We found that the ultrastructure of newly replicated chromatin exhibited a morphology indistinguishable from nucleosomal chromatin. Unreplicated chromatin fibers separating adjacent replicons also exhibit spherical subunits. We inferred that the spherical structures on replicating chromatin are nucleosomes and concluded that histones are not disassociated from the DNA significantly prior to DNA replication, and that a very rapid reassociation of nucleosomes occurs on both daughter DNA molecules following replication. 相似文献
13.
The double-pulse labeling technique for DNA fiber autoradiography was applied to epidermal cells from normal human skin and from human basal cell carcinoma (BCC). We aimed to measure the size and replication rate of the replication unit (RU) for both types of cell and to account, from these results, for our previous observation of a near doubling of S-phase duration in BCC, compared with normal skin. The mean RU size was 76 +/- 4 micron in BCC, not significantly different from the 68 +/- 6 micron value found in normal skin, so the mean of those two values (i.e., 72 micron), was used in further calculations. The rate of replication fork progression was 0.59 +/- 0.005 micron/min in the normal epidermis and 0.33 +/- 0.03 micron/min in BCC, corresponding to a replication time of the average RU equal to 61 min and 109 min, respectively. Thus, with an unchanged RU size in BCC, the observed 1.8-fold decrease in the rate of fork progression in the tumor can account entirely for our previous observation of a 1.8-fold increase in S-phase duration in this tumor, without requiring the assumption of any change in the temporal organization of DNA synthesis in the malignant cells. Considering S phase as an ordered process in which a major part, if not all, of the genome replicates at genetically determined times, we suggest that the clusters of replication units are, in turn, organized into temporally defined "sets". These sets are composed of all the clusters (whatever their chromosomal location) that are programmed to initiate replication during the same fraction of the S period. This hypothesis implies that DNA synthesis in a given set is triggered by some event coupled to progression of replication in the immediately preceding set. Based on a S-phase duration of 10.2 hours in normal skin and of 19.2 hours in BCC (our previous data), and assuming perfect synchrony and homogeneity of the clusters within each set and of each cluster's constitutive RUs, the minimum number of sequentially replicating sets, in both instances, can be estimated as roughly equal to 10. 相似文献
14.
Multiplication of parvovirus LuIII in a synchronized culture system. III. Replication of viral DNA. 总被引:3,自引:3,他引:0
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The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper function(s) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies, and electron microscopy, RF DNA is a linear, double-stranded molecule comparable in length to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Moreover, replication of LuIII DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. It was postulated, therefore, that newly synthesized ssDNA is immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt (1967). 相似文献
15.
M M Zabo?kin V L Moiseev V S Shapot A V Likhtenshte?n 《Molekuliarnaia biologiia》1988,22(4):1119-1127
The two types of DNA-matrix complexes (the weak and tight ones, or type I and type II, respectively) identified in our previous work were studied with respect to their involvement in DNA replication. Nuclei isolated from human fibrosarcoma HT1080 cell line were treated with either restriction endonucleases or ultrasonic desintegrator and afterwards subjected to the triple-gradient Nucleoprotein--Celite chromatography. This permitted fractionation of nuclear DNA into fragments not attached, weakly attached, and tightly attached to the nuclear matrix (DNA 0, DNA I, and DNA II, respectively). It was shown that pulse labelled RNA migrates from DNA II fraction where it resides initially to DNA 0 and further to DNA I during the 2 h chase period. This finding allowed us to consider the tight DNA-matrix complex as the replicative one. The experiments aiming to follow the movements of specific DNA sequences (histone genes) in relation to the DNA-matrix attachment sites were conducted on synchronous HT1080 cells progressing through S phase. The histone sequences appeared to undergo similar movements during the first 30 min of S phase. They reside initially in DNA 0 and DNA I fractions, but as soon as DNA synthesis was restored they migrate consequently to DNA II and DNA 0 fractions. This approach can appear to be a useful tool for studying the schedule of replication of specific genes during S phase. 相似文献
16.
DNA fork displacement rates were measured in Chinese hamster ovary cells (CHO), human HeLa cells and human diploid fibroblasts. For CHO cells two independent techniques were used: one based on CsCl equilibrium density gradients and the other on 313 nm photolysis of incorporated bromodeoxyuridine (BrdUrd). Both methods indicated that there was no significant variation in fork displacement rates in CHO cells as they progressed through S phase. Asynchronous CHO cultures displayed the same average value (1.0 micron/min) and range of values as found in synchronous cells. In contrast, the rate of DNA fork displacement in HeLa cells, measured by the BrdUrd-313 nm method, increased continuously from 0.8 micron/min in early S to 2.5 micron/min in late S. For human diploid fibroblasts, in early S, the rate was approximately 0.7 micron/min and decreased to a minimum of 0.5 micron/min in mid S. The replication fork displacement rate then increased to a maximum of 0.9 micron/min in late S and declined again before the end of S phase. This pattern of DNA fork displacement rates roughly paralleled the overall thymidine incorporation rate and appears quite different from the patterns found for HeLa and CHO cells. 相似文献
17.
Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. 总被引:16,自引:2,他引:14
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The transition from G1 to S phase of the cell cycle may be regulated by modification of proteins which are essential for initiating DNA replication. One of the first events during initiation is to unwind the origin DNA and this requires a single-stranded DNA binding protein. RPA, a highly conserved multi-subunit single-stranded DNA binding protein, was first identified as a cellular protein necessary for the initiation of SV40 DNA replication. The 32 kDa subunit of RPA has been shown to be phosphorylated at the start of S phase. Using SV40 replication as a model, we have reproduced in vitro the S phase-dependent phosphorylation of RPA-32 and show that it occurs specifically within the replication initiation complex. Phosphorylated RPA-32 is predominantly associated with DNA. Phosphorylation is not a pre-requisite for association with DNA, but occurs after RPA binds to single-stranded DNA formed at the origin during the initiation phase. The protein kinase(s) which phosphorylates RPA-32 is present at all stages of the cell cycle but RPA-32 does not bind to the SV40 origin or become phosphorylated in extracts from G1 cells. Therefore, the cell cycle-dependent phosphorylation of RPA-32 may be regulated by its binding to single-stranded origin DNA during replication initiation. 相似文献
18.
The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase 总被引:2,自引:0,他引:2
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Izumi M Yanagi K Mizuno T Yokoi M Kawasaki Y Moon KY Hurwitz J Yatagai F Hanaoka F 《Nucleic acids research》2000,28(23):4769-4777
Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2–7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S.cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G1/S-boundary when quiescent human NB1–RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G2 phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2–7 complexes. 相似文献
19.
The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as “Okazaki” fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4–5 × 105 daltons per min at 26°C, corresponding to the joining of 6–8 Okazaki fragments. The resulting 35–45S (1.1–2.2 × 107 daltons) DNA molecules may correspond to the Physarum “replicon.” The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation. 相似文献
20.
Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci. 总被引:15,自引:8,他引:7
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S M Carroll M L DeRose J L Kolman G H Nonet R E Kelly G M Wahl 《Molecular and cellular biology》1993,13(5):2971-2981
Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus. 相似文献