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At least 1,063 records · Page 59Linked to original sources
PubMed2002

Purification and characterization of the Schizosaccharomyces pombe origin recognition complex: interaction with origin DNA and Cdc18 protein.

The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.

Amino Acid Motifs↗
PubMed1997

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP.

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.

Adenosine Triphosphatases↗
PubMed1987

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells.

Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV-1 cells, permeabilized with lysolecithin, by incubation with [alpha-32P]dTTP, the other dNTPs and rNTPs and an ATP-regenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form II) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent with the involvement of DNA polymerase alpha. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.

Animals↗
PubMed1998

Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins.

Interactions between the herpes simplex virus type 1 (HSV-1) origin (ori)-binding protein (UL9) and two other components of the functional DNA replication complex have been observed. However, to date, no interaction between UL9 and a component of the DNA polymerase holoenzyme has been demonstrated. In this report, we demonstrate that UL9 and the DNA polymerase accessory protein (UL42) can form a stable complex in vitro as determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathione S-transferase (GST) fusion proteins. Complex formation does not require the presence of other viral proteins and occurs in the presence of ethidium bromide, indicating that UL9-UL42 interaction is DNA independent. Affinity beads charged with increasing concentrations of GST-42 fusion protein up to 5 microM bound increasing amounts of UL9 expressed by in vitro transcription/translation in rabbit reticulocyte lysates. Binding of N- and C-terminal portions of UL9 to GST affinity matrices revealed that the N-terminal 533 amino acids were sufficient for binding to GST-42, albeit at approximately a four- to six-fold reduced affinity compared to the full-length protein. No binding of a polypeptide containing the remainder of the UL9 C-terminal residues was observed. Thus the ori-binding protein, UL9, can physically associate with at least one member of each of the complexes (helicase/primase, DNA polymerase holoenzyme, single-stranded DNA-binding protein) required for origin-dependent DNA replication. These specific interactions provide a means by which the ordered assembly of HSV-1 DNA replication proteins at origins of replication can occur in the infected cell for initiation of viral DNA synthesis.

Animals↗
PubMed1990

On the bacterial cell cycle: Escherichia coli mutants with altered ploidy.

We describe a scheme for isolation of new classes of mutants in the cell cycle of Escherichia coli. The mutants were selected as resistant to camphor vapors, which results in increased ploidy, and were subsequently screened for an increase in cell density and an increase in the gene dosage of the lac operon. Our mutations are located at four different places in the chromosome; we have named these loci mbr (moth ball resistant). mbrA maps to 68 min on the E. coli chromosome, mbrB to 88.5 min, mbrC to 89.5 min, and mbrD to 90 min. mbrD mutations may be alleles of rpoB (a subunit of RNA polymerase). In addition to the selected or screened phenotypes, most of the mutants fail to grow on rich media or at high temperatures. We have examined the nine mutants under nonpermissive conditions, using several techniques to determine the cause of death. We have also coupled our mutations with lesions in dnaA, which is required for cell-cycle-specific DNA replication, and rnh (the gene for RNase H), which is required for specificity in the DNA initiation reaction, and determined the effects of the double and triple mutants under permissive and nonpermissive conditions. These tests have shown that bacteria mutated at mbrA do not tolerate a null mutation in rnh, indicating that they are dependent on DNA replication initiating at oriC. In contrast, mutations at mbrB, mbrC, and mbrD exhibit their phenotypes independent of oriC initiation of DNA replication, suggesting that the mutations affect factors that influence the DNA/cell ratio regardless of the origin of DNA replication. Based on our results, the mbr mutations appear to have defects in cell-cycle timing and/or defects in chromosomal partitioning.

Camphor↗
PubMed1986

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen.

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.

Animals↗
PubMed1997

DNA replication of wheat dwarf geminivirus vectors: effects of origin structure and size.

Wheat dwarf virus (WDV) is a representative member of subgroup I of the Geminiviridae, a unique plant DNA virus family. Since geminivirus DNA replication occurs in the host cell nucleus exclusively via double-stranded DNA intermediates, a considerable interest has arisen to use them as expression vectors. We have used particle bombardment to introduce WDV vectors into cultured wheat cells and to analyze the fate of input DNA and the accumulation of newly replicated DNA. Under our conditions, we have found that input DNA, which can be detected immediately after DNA delivery, is rapidly degraded. Newly replicated viral DNA appears approximately 1 day after DNA delivery and reaches a maximum at Days 2-4. Afterward, the total amount of viral DNA is maintained for several days. We have observed a progressive decrease in the relative amount of supercoiled DNA and, concomitantly, an increase in plasmid forms migrating as open circular and nicked DNA. GUS expression from the virion-sense WDV promoter is also maximal 2-3 days after DNA delivery and then it declines to negligible levels 8 days afterward. These results support the conclusion that, under these conditions, reporter gene expression depends on the accumulation of newly replicated, supercoiled plasmid DNA and not on input plasmid DNA. We have also analyzed the effects of WDV origin structure and plasmid size on the accumulation of newly replicated plasmid DNA. Our results lead us to conclude that the replication efficiency of WDV-derived plasmids depends largely on plasmid size. Interestingly, sequences downstream of the initiation site, which in WDV confer an intrinsic curvature to the large intergenic region, seem to have a small effect on the efficiency of plasmid accumulation.

Cells, Cultured↗
PubMed2025

Regulation of replication timing in Saccharomyces cerevisiae.

In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

Saccharomyces cerevisiae↗
PubMed1982

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin, and the effect of DNA replication inhibition.

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize L-[Me-3H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.

5-Methylcytosine↗
PubMed1987

Direct evidence for DNA bending at the lambda replication origin.

Replication initiation in bacteriophage lambda appears to require wrapping of origin DNA on an approximately 50 angstrom radius in or around the complex with the initiator protein O. Since short lengths of DNA are not that flexible, it may be that runs of coherently spaced deoxyadenylate residues constitute bend sites in the ori sequence that facilitate the process. Earlier data showed that ori DNA has electrophoretic anomalies characteristic of bend sites and that these are augmented by initiator protein binding. Here origin bending is examined by direct measurement of the ability of polymerized ori sequences to form small circles. The smallest circles observed (84 residues) are compatible with the required radius of curvature. Bend sites within the O protein binding sites, bend sites in the spacers between them, plus the inherent flexibility of non-bent DNA in the origin may all contribute to origin bending. The data also show that a bend site is required for O protein binding to DNA.

Bacteriophage lambda↗
PubMed1982

Origin of replication in chloroplast DNA of Euglena gracilis located close to the region of variable size.

Chloroplast DNA (cpDNA), containing 10% replicative molecules, was isolated 2 h after onset of the dark period from cultures of Euglena gracilis strain Z. The DNA was digested with the restriction enzymes PvuII, SalI, BamHI, or EcoRI. Fragments that contained intact replicative loops were measured to determine the position of replicated sequences in relation to the restriction enzyme sites. It was found that replication starts at a unique position near one of the palindromic sequences I(2) (Koller and Delius, 1982a) which is located upstream (with respect to the direction of rRNA transcription) of the AT-rich region of variable size (Jenni et al., 1981; Schlunegger et al., in preparation). In the majority of cases DNA synthesis proceeds unidirectionally away from this region for 5000 nucleotides before it starts in the other direction (in the same sense as the rRNA transcription) through the Z-region and the second palindromic sequence.

Journal Article↗
PubMed2002

The origin of DNA genomes and DNA replication proteins.

In recent years, it has became clear that most proteins involved in cellular DNA precursor synthesis or DNA replication have been 'invented' more than once, indicating that the transition from RNA to DNA genomes was more complex than previously thought. Several authors have suggested that DNA viruses, which often encode their own version of these proteins, played an important role in this process. The nature of the genome of the last universal cellular ancestor (LUCA) -- that is, RNA or DNA, prokaryotic-like or eukaryotic-like -- remains in dispute. A hyperthermophilic LUCA would have suggested a circular, double-stranded DNA genome; however, recent data favor a mesophilic or moderately thermophilic LUCA.

Biological Evolution↗
PubMed1979

Origins of phage T4 DNA replication as revealed by hybridization to cloned genes.

[3H]Thymidine-labeled progeny DNA was isolated after infection of Escherichia coli with two different bacteriophage T4 mutants. These strands were isolated shortly after the initiation of DNA replication and hybridized to 15 different (EcoRI) T4 restriction fragments cloned in plasmid vectors. Uniformly labeled T4 [32P]DNA extracted from phage particles was cohybridized as a normalizing reference. The results obtained lead to the conclusions that, among the loci tested, initiation occurs predominantly in the area of genes 50-5 and less prominently in the area of genes 25-29. However, our data do not support the idea of initiation in the area of genes 40-43. In contrast, this area displays the least replication among the genes tested.

Chromosome Mapping↗
PubMed1998

Identification of a protein essential for replication of porcine circovirus.

The largest open reading frame of porcine circovirus (ORF 4) encodes a protein of 312 amino acids. The predicted gene product of ORF 4 shows similarities to Rep proteins of other plant circoviruses and geminiviruses. Three motifs have been identified that are characteristic for proteins involved in rolling circle replication and the consensus sequence for a putative dNTP-binding box (GKS) has been found. In this paper, experimental evidence is presented which indicates that ORF 4 encodes the replication protein of porcine circovirus. After cloning of the ORF 4 gene product, it was supplied in trans in a transient replication assay. The ORF 4 gene product promoted the replication of plasmid pOP11, which carries the origin of DNA replication of porcine circovirus. Since pOP11 itself is unable to replicate in virus-free porcine kidney cells, the ORF 4 gene product must be essential for replication of porcine circovirus.

Amino Acid Sequence↗