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PubMed1991

Replication asynchrony between homologs 15q11.2: cytogenetic evidence for genomic imprinting.

Replication kinetics of the Prader-Willi syndrome critical region (15q11.2) was investigated in seven normal healthy adult females using RBG replication bands. Replication asynchrony between homologs 15q11.2 was identified consistently in about 40% of cells in all individuals. It was limited to the stages in which Xp22, Xp11, Xq13 and Xq24/26 were visible in the late-replicating X chromosome. This asynchrony suggested that replication timing overlapped between 15q11.2 and the early replicating R-bands of the late X chromosome in some cells, and that the difference in replication timing between homologs was probably related to genomic imprinting; the latter has been suggested as a pathogenetic basis of Prader-Willi syndrome. As a result of an analysis of the proportions of asynchronous and synchronous cells in each replication stage, two types of cells were deduced providing 1:1 methylation mosaicism of genomic imprinting was assumed. The first type was composed of cells with normal replication in one homolog and delayed replication in the other. The second type was composed of cells with normal replication in both homologs. Our results provide cytogenetic evidence of methylation mosaicism for mammalian genomic imprinting.

Cell Cycle↗
PubMed1998

Replication timing of the various FMR1 alleles detected by FISH: inferences regarding their transcriptional status.

Following the application of two-color fluorescence in-situ hybridization (FISH) to human interphase cells, we examined the replication timing of the fragile-X locus relative to the non-transcribed late replicating alpha-satellite region of chromosome-X, a built-in intracellular reference locus. In this assay, an unreplicated locus is identified by a single hybridization signal (singlet; S), whereas a replicated locus is identified by a duplicated signal (doublet; D). Hence, following simultaneous hybridization with the FMR1 and alpha-satellite probes, male cells with one singlet and one doublet signal per cell (SD cells) indicate S-phase cells where only one of the two loci has replicated. The studied cell samples (lymphocytes and amniocytes) were derived from normal males, fragile-X male patients, and premutation male carriers. Three distinct populations of SD cells were identified among the various samples. The first population had a high frequency of cells showing a doublet FMR1; this pattern, indicating early replication of FMR1, characterized the SD cell population of normal males. The second population had a high frequency of cells showing a singlet FMR1; this pattern, indicating very late replication of FMR1, characterized the SD population of fragile-X patients. The third population had about one half of the cells showing a singlet FMR1 and the other half with a doublet FMR1, indicating somatic variation in the replication timing of FMR1; this pattern was seen in the SD cell population of premutation carriers. The replication status of the FMR1 locus in the cells of patients was altered from late to early in the presence of 5-azadeoxycytidine, an activator of various silent genes. Based on the vast amount of information showing that expressed loci replicate early, whereas unexpressed loci replicate late, we inferred from the replication status of the FMR1 locus that: (1) the normal FMR1 allele is transcriptionally active in lymphocytes and amniocytes; (2) the fully mutated FMR1 allele is transcriptionally silent; (3) the transcriptional activity of the premutated allele is somewhat disturbed; (4) 5-azadeoxycytidine activates the fully mutated FMR1 allele.

Alleles↗
PubMed1975

Premature replication of late S period DNA regions in early S nuclei transferred to late S cytoplasm by fusion in Physarum polycephalum.

Fusion of a late S period plasmodium of Physarum polycephalum to an early S period plasmodium causes premature replication of late S replicating regions in the nuclei of the early S plasmodium. The extent of ahead-of-schedule replication of late S replicating regions in early S period nuclei increases to a plateau of 16-20% for fusions with 40-70 min of phase difference, then declines for larger phase differences. The stimulatory factors for late S replicative units are present only in late S plasmodia and appear to act only on late S regions. Once replicated, early S replicating regions are not stimulated to replicate again by fusion to a plasmodium entering the S period. Our data do not discriminate between anti-termination of replication by factors of stop sites on long replicons, and a sequential initiation of replication on new, possibly non-adjacent regions, but does provide evidence that the stimulatory factors are distinct from one another and specific for certain target replicative units.

Cell Division↗
PubMed1986

Replication time of interspersed repetitive DNA sequences in hamsters.

The replication time of 34 hamster genomic DNA segments containing interspersed repeat sequences was determined by probing the cloned segments with nick-translated early- and late-replicating hamster DNA. One-third of these cloned families replicated early, one-third replicated late, and one-third replicated without temporal bias. 19 different inserts from these clones along with the SINE, Alu, and the LINE, A36Fc, were used to probe Southern blots of early- and late-replicating hamster or human DNA. We report long interspersed repeats, LINEs, are selectively partitioned into late-replicating DNA and are often concertedly hypomethylated, while short interspersed repeats, SINEs, are selectively partitioned into early-replicating DNA. For some interspersed repeat families, this partitioning is complete or almost complete. The CCGG frequency is very low in late-replicating DNA. The mammalian chromosome's pattern of early-replicating R-bands and late-replicating G-bands reflects a differential distribution of LINEs and SINEs.

Animals↗
PubMed1992

Early and late replicating DNA involved in the G1 to S transition in Allium cepa L meristematic cells.

The involvement of portions of the genome replicated at different times of the S period in the regulation of the G1 to S transition was analyzed in Allium cepa L meristem cells. For this, DNA bromosubstitution confined to discrete portions of a previous S period followed by anoxic UVA irradiation (300-400 nm light) was performed in synchronous cells. Sequences replicated in late S appeared to be involved in the positive regulation of the initiation of replication. Hence, cells were prevented from initiating replication if irradiated at mid G1 only when the DNA sequences replicated in the last third of the previous S period were bromosubstituted. Cycloheximide-induced inhibition of protein synthesis at late G1 also prevented the G1 to S transition. Sequences replicated in mid S appeared unrelated to any control of the initiation of replication. On the other hand, sequences replicated in the first third of the S period seemed to be involved in the negative regulation of the initiation of replication, since irradiation after previous bromosubstitution of early replicating DNA sequences advanced G1 cells into the next S phase and increased the proliferative fraction of the population. Finally, the simultaneous inactivation of DNA sequences involved in both positive and negative regulation of replication allowed the cells to enter into S.

Allium↗
PubMed2000

Bacteriophage and host mutants causing the rolling-circle lambda DNA replication early after infection.

There are two modes of bacteriophage lambda DNA replication during its lytic development in Escherichia coli cells. The circle-to-circle (theta) replication predominates at early stages of the phage growth, whereas rolling-circle (sigma) replication occurs late after infection to produce long concatemers that serve as substrates for packaging of lambda DNA into phage proheads. The mechanism regulating the switch from theta to sigma replication remains unknown. Our previous genetic studies indicated that the bacteriophage lambda Pts1piA66 mutant cannot replicate at 43 degrees C in the wild-type E. coli host, but it can replicate in the dnaA46(ts) mutant. Density shift experiments suggested that the parental DNA molecules of the infecting phage enter sigma replication. Here, using electron microscopy, we demonstrate that as soon as 5 min after infection of the dnaA46(ts) mutant by the lambdaPts1piA66 phage at 43 degrees C, the sigma replication intermediates are highly predominant over theta replication intermediates, contrary to the wild-type conditions (wild-type bacteria infected with the lambdaP(+) phage). The initiation of replication of the lambdaPts1piA66 mutant at 43 degrees C was strongly inhibited in the dnaA(+) host, as demonstrated by electron microscopy and by pulse-labeling of the phage-derived plasmid replicon. Implications for the mechanism of the regulation of the switch from theta to sigma replication mode are discussed.

Bacterial Proteins↗
PubMed2002

Replication timing and transcriptional control: beyond cause and effect.

In general, transcriptionally active euchromatin replicates during the first half of S phase, whereas silent heterochromatin replicates during the second half. Moreover, changes in replication timing accompany key stages of development. Although there is not a strict correlation between replication timing and transcription per se, recent results reveal a strong relationship between heritably repressed chromatin and late replication that is conserved in all eukaryotes. A long-standing question is whether replication timing dictates the structure of chromatin or vice versa. Mounting evidence supports a model in which replication timing is both cause and consequence of chromatin structure by providing a means to inherit chromatin states that, in turn, regulate replication timing in the subsequent cell cycle. Moreover, new findings relating aberrations in replication timing to defects in centromere function, chromosome cohesion and genome instability suggest that the role of replication timing extends beyond its relationship to transcription. Novel systems in both yeasts and mammals are finally beginning to reveal some of the determinants that regulate replication timing, which should pave the way for a long-anticipated molecular dissection of this complex liaison.

Alleles↗
PubMed1996

The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin.

BACKGROUND: The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus. The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc 1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication. Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts. RESULTS: The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MCM-depleted and ORC-depleted extracts restores replication capacity. ORC does not co-localize with sites of DNA replication during elongation. However, at initiation the two staining patterns overlap. In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis. Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin is dependent on the presence of ORC. CONCLUSIONS: The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.

Animals↗
PubMed1999

Protein kinase A is required for chromosomal DNA replication.

Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Analysis of replication timing at the FRA10B and FRA16B fragile site loci.

The molecular basis for the cytogenetic appearance of chromosomal fragile sites is not yet understood. Late replication and further delay of replication at fragile sites expressing alleles has been observed for FRAXA, FRAXE and FRA3B fragile site loci. We analysed the timing of replication at the FRA10B and FRA16B loci to determine whether late replication is a feature which is shared by all fragile sites and, therefore, is a necessary condition for chromosomal fragile site expression. The FRA10B locus was located in a transitional region between early and late zones of replication. Fragile and non-fragile alleles exhibit a similar replication pattern proximal to the repeat but fragile alleles are delayed relative to non-fragile ones on the distal side. Although fragility at FRA10B appears to be caused by expansion of an AT-rich repeat in the region, replication time near the repeat was similar in fragile and non-fragile alleles. The FRA16B locus was late replicating and appeared to replicate even later on fragile chromosomes. While these observations are compatible with the hypothesis that delayed replication may play a role in fragile site expression, they suggest that replication delay may not need to occur at the expanded repeat region itself in order to be permissive for fragility.

Alleles↗
PubMed1999

Interaction of E1 and hSNF5 proteins stimulates replication of human papillomavirus DNA.

Mammalian viruses often use components of the host's cellular DNA replication machinery to carry out replication of their genomes, which enables these viruses to be used as tools for characterizing factors that are involved in cellular DNA replication. The human papillomavirus (HPV) E1 protein is essential for replication of the virus DNA. Here we identify the cellular factor that participates in viral DNA replication by using a two-hybrid assay in the yeast Saccharomyces cerevisiae and E1 protein as bait. Using this assay, we isolated Inil/hSNF5, a component of the SWI/SNF complex which facilitates transcription by altering the structure of chromatin. In vitro binding and immunoprecipitation confirmed that E1 interacts directly with Ini1/hSNF5. Transient DNA-replication assay revealed that HPV DNA replication is stimulated in a dose-dependent manner by addition of Ini1/hSNF5, and that Ini1/hSNF5 antisense RNA blocks the replication of HPV DNA. Amino-acid substitution at residues that are conserved among E1 proteins prevented the E1-Ini1/hSNF5 interaction and reduced DNA replication of HPV in vivo. Our results indicate that Ini1/hSNF5 is required for the efficient replication of papillomavirus DNA and is therefore needed, either alone or in complex with SWI/SNF complex, for mammalian DNA replication as well.

Binding Sites↗
PubMed2000

Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replication.

The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini. Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication. To determine the features required for its unique properties, a deletion analysis was performed on the SGS. For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the 'core' and the flanking sequences to be the 'arms'. The deletion analysis showed that (i) approximately 40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage. These observations implicated the right arm as the unique feature of the SGS. The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication. Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication. The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site. The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication. Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences. Possible roles for the SGS right arm in Mu replication are addressed.

Bacteriophage mu↗
PubMed2004

DNA replication-timing analysis of human chromosome 22 at high resolution and different developmental states.

Duplication of the genome during the S phase of the cell cycle does not occur simultaneously; rather, different sequences are replicated at different times. The replication timing of specific sequences can change during development; however, the determinants of this dynamic process are poorly understood. To gain insights into the contribution of developmental state, genomic sequence, and transcriptional activity to replication timing, we investigated the timing of DNA replication at high resolution along an entire human chromosome (chromosome 22) in two different cell types. The pattern of replication timing was correlated with respect to annotated genes, gene expression, novel transcribed regions of unknown function, sequence composition, and cytological features. We observed that chromosome 22 contains regions of early- and late-replicating domains of 100 kb to 2 Mb, many (but not all) of which are associated with previously described chromosomal bands. In both cell types, expressed sequences are replicated earlier than nontranscribed regions. However, several highly transcribed regions replicate late. Overall, the DNA replication-timing profiles of the two different cell types are remarkably similar, with only nine regions of difference observed. In one case, this difference reflects the differential expression of an annotated gene that resides in this region. Novel transcribed regions with low coding potential exhibit a strong propensity for early DNA replication. Although the cellular function of such transcripts is poorly understood, our results suggest that their activity is linked to the replication-timing program.

Cell Differentiation↗
PubMed1974

Replication of mouse-tropic and xenotropic strains of murine leukemia virus in human x mouse hybrid cells.

The replication of mouse-tropic and xenotropic strains of murine leukemia virus in human x mouse hybrid cells was investigated. NB-tropic strains of the leukemia virus replicated efficiently in several hybrid lines, including those that contained a complete complement of human chromosomes and many mouse chromosomes. In lines with only a few mouse chromosomes, NB-tropic viruses failed to replicate. N- and B-tropic viruses replicated in human x N-type and human x B-type cells, respectively. The N- and B-tropic viruses replicating in these hybrid cells retained their original tropism. The viral restrictive functions of the mouse Fv-1 locus were expressed in the hybrid cells, restricting the replication of N- and B-tropic strains in human x B-type and human x N-type mouse cells, respectively. In contrast to mouse-tropic viruses, AT-124 virus, a xenotropic strain, replicated in human but not in mouse cells or in hybrid cells containing a complete complement of human chromosomes and near complete complement of mouse chromosomes However, hybrid lines with only a few mouse chromosomes supported AT-124 replication. Thus, human genes in hybrid cells do not restrict the replication of mouse or xenotropic murine leukemia virus strains, while mouse genes in such cells restrict xenotropic leukemia virus replication and, as determined by the mouse Fv-1 phenotype, mouse-tropic murine leukemia virus. These results indicate that exogenously applied mouse-tropic and xenotropic oncornaviruses exhibit different patterns of restriction in human-mouse hybrid cells and that such hybrid cells may be used for genetic analysis of oncornavirus replication.

Animals↗
PubMed1986

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analysis in the presence of BrdUrd. Replication of plasmid DNA was observed in the cytoplasm of cells infected with the tumorigenic leporipoxviruses Shope fibroma virus (SFV) and myxoma, and less extensively with the orthopoxvirus vaccinia, but not in uninfected cells. Unexpectedly, all input plasmids tested, including pBR322, pUC13, polyoma, PM2 phi X174 replicative form (RF), and M13 RF, replicated with equal efficiency in SFV-infected cells, indicating that no specific replication origin sequence is required. The transfected plasmid DNA was replicated concomitantly with the infecting poxviral DNA and by 24 hr post-transfection, it resided predominantly in high molecular weight Dpn I-resistant head-to-tail tandem repeats. The failure to detect unreplicated Dpn I-sensitive plasmid concatemers early in replication together with the absence of significant levels of integrated plasmid sequences in the poxviral genome suggest that replication of the transfected plasmid DNA is not the consequence of nonhomologous recombination of concatemeric plasmid DNA into the poxvirus genome, but rather of an autonomous process that is dependent on trans-acting replication factors produced during virus infection, and that does not require a specific origin sequence on the substrate plasmid DNA.

Animals↗
PubMed2004

Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells.

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation.

Cell Culture Techniques↗
PubMed1995

Allele-specific replication timing in imprinted domains: absence of asynchrony at several loci.

Using a bromodeoxyuridine incorporation method to detect replicated DNA, we studied allele-specific replication of several sites within the human Prader-Willi/Angelman and IGF2/H19 imprinted regions. No obvious allele-specific differences in time of replication were detected at most loci previously reported to replicate asynchronously in the same cell types as determined by a FISH-based replication assay. Our finding of an absence of allelic replication asynchrony may be related to low levels of imprinted gene expression near these loci in the examined cells (lymphocytes, fibroblasts and lymphoblastoid cells). This view is supported by our studies of the imprinted SNRPN gene in that cells with paternal allele-specific expression (lymphocytes and lymphoblasts) replicate SNRPN alleles asynchronously, whereas cells with a low level of expression (HeLa) replicate SNRPN later and with less allelic asynchrony. In lymphoblasts, the early replicating allele of SNRPN was identified as the paternal one based on the properties of maternal allele-specific methylation and paternal allele-specific expression. Our studies suggest that FISH data implying replication asynchrony in nonexpressing cells reflect structural differences between the maternal and paternal alleles rather than differences in replication timing.

Alleles↗
PubMed1995

Reverse replication timing for the XIST gene in human fibroblasts.

The timing of DNA replication appears to be an important epigenetic regulator of gene expression during development. Replication of active genes in expressing tissues occurs earlier than does replication of their inactive counterparts in nonexpressing tissues. This pattern is also observed for active and inactive alleles present in the same cell, as exemplified by genes subject to X chromosome inactivation in females. We find that the replication timing of the X-linked XIST gene in normal human fibroblasts provides a striking exception to this well-established pattern. Within the same cell, the expressed allele of XIST replicates late in S phase and the silent allele replicates early. This 'reverse' replication timing may have functional significance with respect to XIST or could be a passive consequence of the replication timing requirements of neighboring genes that are subject to X chromosome inactivation. Our finding of early replication for XIST in male fibroblasts contrasts with a report of late replication in such cells as determined by an in situ hybridization method [Torchia et al., (1994) Am. J. Hum. Genet. 55, 96-104]. We propose that our data and those obtained by the in situ method can be accommodated by the existence of structural features that differ between the silent and expressed alleles of XIST. Similar features may be important determinants of the replication asynchrony found by the in situ method for other genes subject to monoallelic expression.

Alleles↗