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At least 163 records · Page 9Linked to original sources
PubMed1988

Studies on early replication patterns in the marsupial Sminthopsis crassicaudata: no evidence for XY replication homologies.

We present here the first detailed replication banding study of a marsupial species using the BrdU-replication technique. A comparison of the structural and replication bands of the chromosomes of Sminthopsis crassicaudata clearly demonstrates that the replication behavior is the same as the described for the chromosomes of eutherians. The early replicating segments correspond to R-bands, whereas the late-replicating regions tend to be situated within Q- and C-bands. Use of this technique clearly reveals an early and late replicating X chromosome. The very small Y chromosome can be subdivided into two replication segments, but no replication homologies can be demonstrated between the X and Y chromosomes of S. crassicaudata.

Animals↗
PubMed1992

Patterns of DNA replication in Drosophila polytene nuclei replicating in Xenopus egg and oocyte extracts.

We have used Xenopus laevis cell-free extracts to study patterns of DNA replication in polytene nuclei isolated from salivary glands of Drosophila melanogaster 3rd instar larvae. Replication was visualized by supplementation with biotin-dUTP so that nascent DNA became labelled, thus allowing detection with fluorescein or Texas-Red-conjugated streptavidin. Biotin incorporation was dependent on incubation in extracts. Transverse bands were labelled in high-speed supernatants of eggs or oocytes in which replication does not initiate de novo. These patterns corresponded to the patterns of endogenous replication forks in polytene nuclei, monitored by bromodeoxyuridine incorporation in intact salivary glands. By contrast, when nuclei were incubated in low-speed supernatants of eggs, they underwent more extensive chromatin decondensation and initiated replication. The spatial patterns of replication are strikingly different from the endogenous patterns. Instead they closely resemble patterns of clustered replication forks seen in Xenopus sperm nuclei replicating in the extract. This indicates that the egg extract can impose its pattern of replication foci even when the template is presented in the highly organized form of a polytene nucleus.

Animals↗
PubMed1996

Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication.

Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.

Animals↗
PubMed1980

Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system.

A wide variety of double-stranded DNA templates are replicated extensively in an in vitro DNA replication system containing the purified proteins specified by seven T4 bacteriophage DNA replication genes (32, 41, 43, 44, 62, 45, and 61). In favorable conditions, this multiprotein system catalyzes the synthesis of several copies of the input DNA template in a 30- to 60-min incubation. The replication forks produced in vitro move in a highly processive fashion, at approximately the in vivo rate of 500 nucleotides per s. The DNA synthesized on the lagging side of the in vitro replication fork is made discontinuously, as it is in vivo, giving rise to "Okazaki pieces" averaging some 10,000 nucleotides in length; in contrast, DNA is polymerized in a continuous manner on the leading side of the in vitro fork. Although the mechanism by which the seven-protein in vitro DNA replication system propagates replication forks closely resembles the in vivo mechanism, it lacks the capacity to remove RNA primers, to reseal Okazaki pieces, and to initiate replication forks at defined DNA origins; supplementation of the system with additional T4-specific replication proteins will be required to facilitate these latter three functions.

DNA, Viral↗
PubMed1993

Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria.

In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria. In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions. Specifically, TrfA proteins with deletions or substitutions of the terminal cysteine, lysine, and arginine (codons 380-382, respectively) were constructed and characterized for their ability to initiate replication from an RK2 origin in vivo in E. coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the iterons at the replication origin. Substitutions of the cysteine at position 380 with a glycine or an arginine resulted in a TrfA protein defective in binding to the RK2 origin and, therefore, defective in replication initiation activity in all four Gram-negative bacteria. Substitution of a serine at that position preserved limited function in replication and DNA binding. The lysine at position 381 could be changed to a glutamine without any obvious change in TrfA function. Deletion of the terminal arginine at position 382 did not affect the ability of TrfA to bind to origin iterons but caused a complete loss of replication activity in all four bacteria. Substitution of this terminal arginine with alanine, serine, or glutamic acid also produced replication-defective TrfA protein in all four bacterial hosts while not affecting iteron binding activity. However, substitution of this arginine with a lysine resulted in a loss of replication activity in E. coli and A. vinelandii but had no effect in P. putida and A. tumefaciens. These observations suggest that the terminal arginine plays an essential role in the activity of the TrfA protein, possibly interaction with host proteins, which can be separated from its iteron binding activity.

Amino Acids↗
PubMed1995

Stimulation of DNA synthesis by mouse DNA helicase B in a DNA replication system containing eukaryotic replication origins.

A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far. We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin. In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin. We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen. DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase. DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A. A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B. Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system.

Adenosine Triphosphatases↗
PubMed2001

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.

Animals↗
PubMed1983

Studies on the replication of Escherichia coli phage lambda DNA. I. The kinetics of DNA replication and requirements for the generation of rolling circles.

Escherichia coli phage lambda DNA has been isolated from infected bacteria using a new technique by which virtually all phage DNA is recovered. Isolated DNA is examined by electron microscopy. Addition of phi X174 RF1 molecules as a counting standard enables us to determine the average number of lambda DNA molecules present in an infected cell. In this study, we have followed the kinetics of lambda DNA replication and examined rolling circle replication. The most important findings are the following: (1) Rolling circle replication is initiated at roughly the same time as is theta replication, indicating that the rolling circle is not solely a late-replicating form. (2) theta replication stops at about 16 min after infection. (3) Early in infection the number of DNA molecules per cell doubles every 2-3 min until theta replication stops, at which point most DNA synthesis consists of growth of the tails of about three rolling circles per cell. (4) Neither the timing of rolling circle replication nor the number of molecules is affected by the activity of the lambda red genes. (5). The red genes are responsible for the production of oligomeric circles late in infection.

Bacteriophage lambda↗
PubMed1981

The remarkable instability of replication loops provides a general method for the isolation of origins of DNA replication.

Closed superhelical replicative intermediates of SV40 DNA were stable during incubation at elevated temperatures for prolonged times. However, when the replicative intermediates were nicked or made linear by digestion either outside or inside of the replication loop, the nascent DNA strands were spontaneously extruded at a measurable rate even at 37 degrees C. The extruded DNA was double-stranded. The rate-limiting step in the extrusion was not the rate of branch migration. The instability of replication loops suggested a method for the isolation of replication origins. The method has been tested by a reconstruction experiment in which a small amount of linear SV40 replicative intermediates was mixed with a vast excess of cellular DNA. The mixture was heated overnight at 55 degrees C, and the spontaneously extruded nascent strands were isolated and inserted into pBR322. Plasmids containing SV40 DNA of less than genomic size, symmetrically distributed about the SV40 replication origin, were isolated. This result suggests that the method is generally applicable to the isolation of replication origins.

DNA Replication↗
PubMed2002

The amino-terminus of the E2F-1 transcription factor inhibits DNA replication of autonomously replicating plasmids in mammalian cells.

The E2F1 transcription factor plays a pivotal role in driving cells out of a quiescent state and into the S phase of the cell cycle, in part by transactivating genes needed for DNA replication including DHFR, thymidine kinase, and DNA Polymerase alpha. E2F1 has also been implicated in regulating an S phase checkpoint, however its role in this checkpoint is not well defined. To determine how E2F1 affects such a checkpoint, we utilized an in vivo replication assay employing a plasmid based SV40 origin of replication, transfected into cells expressing SV40 large T antigen. Here we show that expression of full length E2F1, or only its N terminus, represses replication from plasmids containing the SV40 origin, while N terminal deletions of E2F1 do not. E2F1 appears to inhibit the elongation phase of replication and not the initiation phase since it does not affect the replication of other cotransfected plasmids containing only the SV40 origin. Further, inhibition of replication is dependent on both the amino-terminus of the E2F1 protein and on a DNA sequence that is contained within the 3' end of the E2F1 cDNA. Additionally, both full-length E2F1, or just its N-terminus, form protein complexes with two portions of the 3' end of the E2F1 cDNA. These data provide a clue to the mechanism by which E2F1 regulates transit through the S phase checkpoint, by acting on a specific DNA sequence via its amino-terminal region, to inhibit elongation of DNA replication.

Animals↗
PubMed2003

Titration of the Escherichia coli DnaA protein to excess datA sites causes destabilization of replication forks, delayed replication initiation and delayed cell division.

In Escherichia coli, the level of the initiator protein DnaA is limiting for initiation of replication at oriC. A high-affinity binding site for DnaA, datA, plays an important role. Here, the effect of extra datA sites was studied. A moderate increase in datA dosage ( approximately fourfold) delayed initiation of replication and cell division, but increased the rate of replication fork movement about twofold. At a further increase in the datA gene dosage, the SOS response was induced, and incomplete rounds of chromosome replication were detected. Overexpression of DnaA protein suppressed the SOS response and restored normal replication timing and rate of fork movement. In the presence of extra datA sites, cells showed a dependency on PriA and RecA proteins, indicating instability of the replication fork. The results suggest that wild-type replication fork progression normally includes controlled pausing, and that this is a prerequisite for normal replication fork function.

Adenosine Triphosphatases↗
PubMed2004

Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination.

Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using microarray-based marker frequency analysis. Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms. Two of the three replication origins in each species were located in the vicinity of a cdc6/orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism. In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated. In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic elongation rates. In S. acidocaldarius, a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the chromosome, raising the possibility of a centromere-like function.

Chromosomes↗
PubMed2005

Replication-associated strand asymmetries in mammalian genomes: toward detection of replication origins.

In the course of evolution, mutations do not affect both strands of genomic DNA equally. This imbalance mainly results from asymmetric DNA mutation and repair processes associated with replication and transcription. In prokaryotes, prevalence of G over C and T over A is frequently observed in the leading strand. The sign of the resulting TA and GC skews changes abruptly when crossing replication-origin and termination sites, producing characteristic step-like transitions. In mammals, transcription-coupled skews have been detected, but so far, no bias has been associated with replication. Here, analysis of intergenic and transcribed regions flanking experimentally identified human replication origins and the corresponding mouse and dog homologous regions demonstrates the existence of compositional strand asymmetries associated with replication. Multiscale analysis of human genome skew profiles reveals numerous transitions that allow us to identify a set of 1,000 putative replication initiation zones. Around these putative origins, the skew profile displays a characteristic jagged pattern also observed in mouse and dog genomes. We therefore propose that in mammalian cells, replication termination sites are randomly distributed between adjacent origins. Taken together, these analyses constitute a step toward genome-wide studies of replication mechanisms.

Animals↗