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At least 19 recordsLinked to original sources
PubMed1994

Minichromosome replication in vitro: inhibition of re-replication by replicatively assembled nucleosomes.

Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.

Centrifugation↗
PubMed2000

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.

Animals↗
PubMed1996

Initiation of baculovirus DNA replication: early promoter regions can function as infection-dependent replicating sequences in a plasmid-based replication assay.

From the results of transient plasmid-based replication assays, it has been postulated that homologous regions (hrs) of Autographa californica nuclear polyhedrosis virus (AcMNPV) function as origins of viral DNA replication. However, these assays vary in specificity according to the methodology used and may not be dependent solely on the presence of hr sequences. To determine the role that hrs and other sequences might play in the replication process, a series of plasmids containing specific deletions of various hrs was generated and tested in a standardized replication assay. Deletion of the AcMNPV hr2 and hr5 sequences abolished the ability of plasmids to replicate in the standard infection-dependent replication assay, while deletion of hr1, hr3, and hr4a sequences decreased but did not eliminate plasmid replication in this assay. Plasmids carrying the complete ie-2 and pe38 genes, the ie-1 gene upstream region, or a variety of baculovirus genes including 11 early promoter regions were also able to replicate in virus-infected cells, suggesting that early viral promoter sequences could also function as putative origins of replication. These data suggest that the standard infection-dependent replication assay may identify a broad range of infection-dependent replicating sequences, only one or a few of which may represent genuine viral origins used by the virus in vivo. We propose a model suggesting that the selection of replication initiation sites may be imposed directly by chromatin structure and indirectly by primary sequence and that the process of viral DNA replication may be linked with viral transcription.

Baculoviridae↗
PubMed1995

S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state.

BACKGROUND: DNA replication and mitosis are triggered by activation of kinase complexes, each made up of a cyclin and a cyclin-dependent kinase (Cdk). It had seemed possible that the association of Cdks with different classes of cyclins specifies whether S phase (replication) or M phase (mitosis) will occur. The recent finding that individual B-type cyclins (encoded by the genes CLB1-CLB6) can have functions in both processes in the budding yeast Saccharomyces cerevisiae casts doubt on this notion. RESULTS: S. cerevisiae strains lacking C1b1-C1b4 undergo DNA replication once but fail to enter mitosis. We have isolated mutations in two genes, SIM1 and SIM2 (SIM2 is identical to SEC72), which allow such cells to undergo an extra round of DNA replication without mitosis. The Clb5 kinase, which promotes S phase, remains active during the G2-phase arrest of cells of the parental strain, but its activity declines rapidly in sim mutants. Increased expression of the CLB5 gene prevents re-replication. Thus, a cyclin B-kinase that promotes DNA replication in G1-phase cells can prevent re-replication in G2-phase cells. Inactivation of C1b kinases by expression of the specific C1b-Cdk1 inhibitor p40SIC1 is sufficient to induce a prereplicative state at origins of replication in cells blocked in G2/M phase by nocodazole. Re-activation of C1b-Cdk1 kinases induces a second round of DNA replication. CONCLUSIONS: We propose that S-phase-promoting cyclin B--Cdk complexes prevent re-replication during S, G2 and M phases by inhibiting the transition of replication origins to a pre-replicative state. This model can explain both why origins 'fire' only once per S phase and why S phase is dependent on completion of the preceding M phase.

Cell Cycle↗
PubMed1982

Patterns of DNA replication of human chromosomes. II. Replication map and replication model.

Combining higher resolution chromosome analysis and bromodeoxyuridine (BrdU) incorporation, our study demonstrates that: (1) Human chromosomes synthesize DNA in a segmental but highly coordinated fashion. Each chromosome replicates according to its innate pattern of chromosome structure (banding). (2) R-positive bands are demonstrated as the initiation sites of DNA synthesis in all human chromosomes, including late-replicating chromosomes such as the LX and Y. (3) Replication is clearly biphasic in the sense that late-replicating elements, such as G-bands, the Yh, C-bands, and the entire LX, initiate replication after it has been completed in the autosomal R-bands (euchromatin) with minimal or no overlap. The chronological priority of R-band replication followed by G-bands is also retained in the facultative heterochromatin or late-replicating X chromosome (LX). Therefore, the inclusion of G-bands as a truly late-replicating chromatin type or G(Q)-heterochromatin is suggested. (4) Lateral asymmetry (LA) in the Y chromosome can be detected after less than half-cycle in 5-bromodeoxyuridine (BrdUrd), and the presence of at least two regions of LA in this chromosome is confirmed. (5) Finally, the replicational map of human chromosomes is presented, and a model of replication chronology is suggested. Based on this model, a system of nomenclature is proposed to place individual mitoses (or chromosomes) within S-phase, according to their pattern of replication banding. Potential applications of this methodology in clinical and theoretical cytogenetics are suggested.

Bromodeoxyuridine↗
PubMed1989

Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro.

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.

Adenosine Triphosphatases↗
PubMed2006

Epstein-Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments.

Epstein-Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain-enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

Cell Line, Tumor↗
PubMed1993

Replication of minute virus of mice minigenomes: novel replication elements required for MVM DNA replication.

In this report, we describe the replication of minigenomes of minute virus of mice (MVM). We show that the cis-acting sequences required for MVM DNA replication reside in the terminal 140 and 660 nucleotides of the left and right termini, respectively. Minigenomes containing either two right (RR) or two left (LL) termini are replication competent genomes, demonstrating that both termini contain the genetic information necessary for the excision and initiation of DNA replication. Since the efficiency of replication of the RR genome is greater than that of the LL genome, it suggests that the individual terminal sequences are not equivalent in function. In addition to the terminal palindromic sequences required for replication, we show that specific elements found inboard of the right hairpin between nucleotides 4489-4636 (element A) and 4636-4695 (element B) are necessary for the efficient replication of MVM minigenomes. These elements have heretofore not been identified as replication sequences.

Animals↗
PubMed1987

Bacteriophage P4 DNA replication. Nucleotide sequence of the P4 replication gene and the cis replication region.

A 3100 base piece of DNA from the 11,500 base genome of bacteriophage P4 was analyzed for its nucleotide sequence. This segment of DNA contains two open reading frames of 106 and 777 amino acid residues; the latter of which is the coding sequence for the Mr 84,841 alpha protein, which is necessary for P4 DNA replication and is thought to act as a P4-specific DNA primase. A region of about 300 base-pairs localized just beyond the alpha gene and about 4500 bases from the origin of replication (ori), was defined as the locus for P4's cis replication region (crr). This region is required for replication both in vivo and in vitro, and consists of two directly repeated sequences of 120 base-pairs that match one another at 98 positions. These directly repeated sequences are separated by 60 base-pairs, which are not necessary for replication. Each repeat in crr contains three copies of the octamer TGTTCACC that is found six times in ori. Either of the 120 base-pair repeat sequences in crr is sufficient for replication, and the entire crr can function in an inverted orientation. crr is also active at a distance of 1800 bases from the P4 origin of replication.

Bacteriophages↗
PubMed1991

Iteron inhibition of plasmid RK2 replication in vitro: evidence for intermolecular coupling of replication origins as a mechanism for RK2 replication control.

The broad-host-range plasmid RK2 and its derivatives are maintained in Gram-negative bacteria at a specific copy number that appears to be determined by a series of direct repeats (iterons) located at the RK2 replication origin and by the RK2 replication initiation protein. TrfA. An in vitro replication system was developed from Escherichia coli that is active with either the intact eight-iteron RK2 origin or a minimal five-iteron RK2 origin when purified TrfA protein is provided. Using this in vitro replication system, we have examined the mechanism(s) of copy-number control. It was found that two or more RK2 iterons present on a supercoiled compatible plasmid molecule are capable of specifically inhibiting in vitro the replication of either functional RK2 origin plasmid and that this inhibition is not overcome by adding increasing amounts of TrfA protein. A mutant TrfA protein, TrfA-33(cop254D), that increases the copy number of an RK2 origin in vivo exhibits replication kinetics and activity levels in this in vitro system similar to that of the wild-type protein. However, RK2 in vitro replication initiated by TrfA-33(cop254D) has a much reduced sensitivity to iteron inhibition. These data support a model for RK2 copy-number control that involves intermolecular coupling between TrfA-bound iterons.

Bacterial Proteins↗
PubMed1984

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome.

The replication origin (ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [3H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a lambda Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels. Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.

Bacillus subtilis↗
PubMed1994

Replicative intermediates of human papillomavirus type 11 in laryngeal papillomas: site of replication initiation and direction of replication.

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180 degrees opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

DNA Replication↗
PubMed1999

Replication factor C3 of Schizosaccharomyces pombe, a small subunit of replication factor C complex, plays a role in both replication and damage checkpoints.

We report here the isolation and functional analysis of the rfc3(+) gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3(+) gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3(+) is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

Base Sequence↗
PubMed1980

Functional analysis of minichromosome replication: bidirectional and unidirectional replication from the Escherichia coli replication origin, oriC.

Replicating molecules of minichromosomes pCM959 and pOC24 were analyzed by electron microscopy. Replication of pCM959 proceeded bidirectionally from the replication origin, oriC, in about 60% of the molecules; the rest of the molecules replicated unidirectionally in either direction. pOC24, in which deoxyribonucleic acid to the right (clockwise) of the oriC segment is deleted, seemed to replicate predominantly unidirectionally counterclockwise from oriC.

Base Sequence↗
PubMed1980

Control of plasmid R1 replication: functions involved in replication, copy number control, incompatibility, and switch-off of replication.

A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42 degrees C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42 degrees C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 x 10(6)), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.

Chromosomes, Bacterial↗
PubMed2005

Specific binding of tombusvirus replication protein p33 to an internal replication element in the viral RNA is essential for replication.

The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus, Tomato bushy stunt virus (TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C.C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related Turnip crinkle virus and distantly related Hepatitis C virus to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in Saccharomyces cerevisiae cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in Tombusviridae encode comparable replicase proteins, these results may be relevant to other members of this large virus family.

Binding Sites↗