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PubMed1992

Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication.

The mechanism by which X rays inhibit DNA replication has been investigated in three distinct populations of DNA molecules in human cells: (a) large chromosomal DNA, (b) a population of 50-100 10.3-kb nuclear episomal plasmids per cell, and (c) a population of about 500 16-kb cytoplasmic mitochondrial DNA molecules per cell. DNA replication was inhibited by X rays in nuclear chromosomal and plasmid DNA, but not in mitochondrial DNA. The mechanism by which ionizing radiation inhibits DNA replication must therefore be nuclear-specific and is unlikely to involve diffusible low-molecular-weight substances. Since mitochondrial DNA exists in the cell as independent 16-kb circular molecules and responds to radiation as would be expected for small targets, the implication for nuclear plasmids is that their replication is regulated by a large target. A current model for DNA replication involves the movement of DNA through replication centers made up of polymerases, helicases, and associated replication enzymes that are attached to a matrix. The difference in the response to X rays between mitochondrial DNA and nuclear plasmid DNA can be explained if nuclear plasmids are tightly associated with chromosomal DNA and attached to the matrix, and are coordinately replicated.

DNA Replication↗

PubMed1990

Host virus interactions in the initiation of bacteriophage lambda DNA replication. Recruitment of Escherichia coli DnaB helicase by lambda P replication protein.

The bacteriophage lambda P protein promoters replication of the phage chromosome by recruiting a key component of the cellular replication machinery to the viral origin. Specifically, P protein delivers one or more molecules of Escherichia coli DnaB helicase to a nucleoprotein structure formed by the lambda O initiator at the lambda replication origin. Using purified proteins, we have examined the features of the pivotal host virus interaction between P and DnaB. These two proteins interact in vitro to form a P.DnaB protein complex that can be resolved by sedimentation or by chromatography on DEAE-cellulose from the individual free proteins. The sedimentation coefficient of the P.DnaB complex, 13 S, suggests a size larger than that of free DnaB hexamer (Mr = 313,600). The P.DnaB complex isolated by glycerol gradient sedimentation contains approximately three protomers of P/DnaB hexamer, consistent with a molecular weight of 393,000. The isolated P.DnaB complex functions in vitro in the initiation of lambda DNA replication. Interaction of P with DnaB strongly suppressed both the intrinsic DNA-dependent ATPase activity of DnaB, as well as the capacity of DnaB to assist E. coli primase in the general priming reaction. Formation of a P.DnaB protein complex also blocked DnaB from functioning in the initiation of E. coli DNA replication in vitro. The physical and functional properties of lambda P protein suggest that it is a viral analogue of the E. coli DnaC replication protein. Like P, DnaC also binds to DnaB (Wickner, S., and Hurwitz, J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925), but unlike P, DnaC stimulates DnaB-mediated general priming. When viral P and bacterial DnaC replication proteins were placed in direct competition with one another for binding to DnaB, the viral protein was clearly predominant. For example, a 5-fold molar excess of DnaC protein only partially reversed the inhibitory effect of P on general priming. Furthermore, when a preformed DnaC.DnaB protein complex was incubated briefly with P protein, it was readily converted into a P.DnaB protein complex and the bulk of the bound DnaC was released as free protein. It is likely that the capacity of the lambda P protein to outcompete the analogous host protein for binding to the bacterial DnaB helicase is the critical molecular event enabling infecting phage to recruit cellular replication proteins required for initiation of DNA synthesis at the viral origin.

Bacterial Proteins↗

PubMed1985

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.

Bacterial Proteins↗

PubMed1989

Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.

Antigens, Polyomavirus Transforming↗

PubMed1992

Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A.

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

Amino Acid Sequence↗

PubMed1987

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication.

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbilllus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially "reinactivated", suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.

Animals↗

PubMed1988

Replication of ColE2 and ColE3 plasmids: the regions sufficient for autonomous replication.

We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions. A 1.3 kb segment of each plasmid is sufficient for autonomous replication. Plasmids carrying this segment retain the replication properties of the original plasmid. The 1.3 kb segment consists of three functional portions. Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid. Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter. The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid. The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s). The action of the trans-acting factor(s) on the origin is plasmid specific. The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.

Colicins↗

PubMed1987

E. coli minichromosome replication in vitro and in vivo: comparative analyses of replication intermediates.

The process of replication of Escherichia coli minichromosomes was examined by following the intermediates formed in vitro and in vivo. Replication initiated on a supercoiled closed circular (CC) monomer, proceeded rapidly to a late but incomplete stage in polymerization (the LC form) in both systems, passed more slowly through a series of open and closed circular catenated dimers with varying extents of intertwining between the monomer units, and then yielded, after decatenation, the supercoiled CC monomer. The replication patterns of two different minichromosomes were similar, although the LC form and the multiply intertwined dimers were much more evident in the smaller pAL4 than in pAL2. The same basic replication scheme was seen in vitro and in vivo but completion of polymerization and processing of the dimers were slower in vitro. Some radioactivity was detected in OC monomer early during replication, consistent with occasional decatenation of LC structures to produce OC molecules which then completed replication to form CC molecules. However, progression to CC catenated dimers prior to formation of CC monomers represented the major replication pathway.

Centrifugation, Density Gradient↗

PubMed1972

Relationship of Flac replication and chromosome replication.

The time of replication of a bacterial plasmid, Flac, during the division cycle of Escherichia coli has been estimated in exponentially growing cultures and at various times after a shift from minimal medium to a richer medium (a shift-up). There is a variation in the cell age at which the capacity to synthesize beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) doubles (assumed to be a measure of the time at which the Flac plasmid replicates) when this capacity is measured at various times during the shift-up, and with increasing steady-state exponential growth rate. Cells growing at slow and moderate growth rates exhibit Flac replication in the middle of the division cycle. With increasing time after a shift-up or with increasing growth rate the plasmid replicates at earlier times, eventually at cell division, and finally in the older cells. This variation in the cell age at which the plasmid replicates is similar to the variation in cell age at which chromosome initiation occurs during a shift-up, although plasmid replication occurs slightly before initiation of chromosome replication.

Amino Acids↗

PubMed1974

Replication of colicin E1 plasmid DNA in minicells from a unique replication initiation site.

Replicating DNA molecules of the colicin E1 plasmid isolated from minicells are cleaved at a single site by R1 restriction endonuclease (EcoR1). Electron microscopic measurements of the replicating molecules treated with the endonuclease indicate that (a) replication is initiated at a site between 14% and 20% of the distance from the EcoR1 endonuclease cleavage site; and (b) extensive replication of most molecules occurs in one direction from the initiation site, although a limited amount of replication in the opposite direction may occur. Single-stranded regions at one or both replication forks, involving one or both DNA strands, can be frequently found in replicating molecules.

Colicins↗

PubMed1990

The monomer covalently closed linear replicative form DNA is an intermediate of Aleutian disease parvovirus DNA replication.

In this report we present data indicating that the recently detected monomer covalently closed linear replicative form DNA (Mccl RF DNA) is an intermediate of Aleutian disease virus (ADV) DNA replication. This DNA molecule is characterized by covalently closed terminal hairpins on either end of the linear ds genomic molecule. Its first detection early after infection in vitro, the association with ADV-specific replication complexes and the de novo synthesis of Mccl RF DNA by isolated replication complexes point to an important role in parvoviral DNA replication. The presence of the Mccl RF DNA in extracts of virus-producing bone marrow cells of naturally infected mink rules out the possibility that this DNA form represents a mere cell culture artefact. The detection of the ADV non-structural protein 1 (NS1) in replication complexes supports the view that this protein is involved in ADV DNA replication.

Aleutian Mink Disease Virus↗

PubMed1974

Replication of the bacteriocinogenic plasmid Clo DF13 in thermosensitive Escherichia coli mutants defective in initiation or elongation of deoxyribonucleic acid replication.

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.

Bacterial Proteins↗

PubMed1987

Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BglII-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants after the transfection of p65-tk into mouse FM3Atk- cells. The copy numbers of p65-tk in FM3Atk+ cells were 100 to 200 copies per cell. Furthermore, the p65-tk replicated semiconservatively, and the initiation of DNA replication started from the mouse DNA sequences when the replicating activity of p65-tk was tested in the in vitro DNA replication system developed from the FM3A cells. These results show that a 2.5-kilobase fragment of mouse DNA contains the autonomously replicating sequences.

Animals↗

PubMed1991

Cytokine influence on simian immunodeficiency virus replication within primary macrophages. TNF-alpha, but not GMCSF, enhances viral replication on a per-cell basis.

The control of HIV-1 or SIV replication within macrophages is probably influenced by a variety of viral and cellular factors. Of the cellular factors, the authors have studied cytokine influence on SIV replication in vitro utilizing simian alveolar macrophages and uncloned SIVmacMTV, a macrophage-tropic variant. The approach allowed quantification of viral replication on a per-cell basis. As reported for HIV-1 replication in macrophages, TNF-alpha significantly increased SIV production in these macrophage cultures. GMCSF also resulted in marked increases in SIV gag protein in culture supernatants. However, after correcting for differences in total cell numbers and numbers of gag-containing cells in the treated and untreated cultures, GMCSF did not upregulate SIV production on a per-cell basis. IL-6 increased SIV replication little if at all but induced significantly greater cytopathic changes in the treated cultures compared with infected, untreated cultures. In contrast, IFN-gamma greatly decreased replication. Our results for GMCSF, IFN-gamma, and IL-6 are in contrast to previously published reports of cytokine control of HIV-1 growth in target cells, and they stress the importance of cell number analyses and the use of primary cultures in the study of lentiviral replication kinetics in macrophages.

Animals↗

PubMed2025

Nanopore-based sequencing of active DNA replication reveals key principles of metazoan replication fork progression, origin and termination sites.

Balancing replication fork progression and origin usage is essential to maintain genome stability, but measuring replication fork progression rates and origin usage throughout the genome has been challenging. Here, we use nanopore sequencing combined with DNAscent to measure replication fork progression together with origin and termination site usage with single-molecule precision throughout the Drosophila genome with nearly full genome coverage. We find that replication fork progression rates are not uniform throughout the genome. Rather, fork progression is slowest in euchromatin, and this is not correlated with active transcription. Replication origins are also influenced by chromatin, but the exact position of initiation is highly variable and are often several kilobases away from ORC binding sites. Termination sites lack any chromatin or sequence motifs and appear nearly random throughout the genome. By measuring DNA replication dynamics at near full genome coverage, our work reveals key principles of metazoan replication dynamics.

Journal Article↗

PubMed1986

Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures.

The structure of replicating simian virus 40 (SV40) minichromosomes was studied by DNA crosslinking with trimethyl-psoralen. The procedure was used both in vitro with extracted SV40 minichromosomes as well as in vivo with SV40-infected cells. Both procedures gave essentially the same results. Mature SV40 minichromosomes are estimated to contain about 27 nucleosomes (error +/- 2), except for those molecules with a nucleosome-free gap, which are interpreted to contain 25 nucleosomes (error +/- 2). In replicative intermediates, nucleosomes are present in the unreplicated parental stem with the replication fork possibly penetrating into the nucleosomal DNA before the histone octamer is removed. Nucleosomes reassociate on the newly replicated DNA branches at distances from the branch point of 225 ( +/- 145) nucleotides on the leading strand and of 285( +/- 120) nucleotides on the lagging strand. In the presence of cycloheximide, daughter duplexes contained unequal numbers of nucleosomes, supporting dispersive and random segregation of parental nucleosomes. These were arranged in clusters with normal nucleosome spacing. We detected a novel type of interlocked dimer comprising two fully replicated molecules connected by a single-stranded DNA bridge. We cannot decide whether these dimers represent hemicatenanes or whether the two circles are joined by a Holliday-type structure. The joining site maps within the replication terminus. We propose that these dimers represent molecules engaged in strand segregation.

Cross-Linking Reagents↗

PubMed1989

The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication.

Epstein-Barr virus (EBV) oriP contains two components, a dyad symmetry element and a direct repeat element, that, in the presence of EBV nuclear antigen 1, are necessary and sufficient for plasmid replication. We have examined the replicative forms generated by EBV oriP using 2D gel electrophoresis. The patterns obtained from an oriP plasmid in a transfected cell line indicate that the site of initiation of DNA replication is at or very near the dyad symmetry element, while the direct repeats contain a replication fork barrier and the termination site. Thus, replication from oriP proceeds in a predominantly undirectional manner. The patterns obtained from cells immortalized by EBV suggest that replication from oriP proceeds similarly in the viral genome.

DNA Replication↗

PubMed1991

Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.

Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).

Adenosine Triphosphate↗