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PubMed1992

EBNA1 distorts oriP, the Epstein-Barr virus latent replication origin.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed.

Antigens, Viral↗
PubMed1984

Cloning and delimiting one chloroplast DNA replicative origin of Chlamydomonas.

The EcoR1 restriction fragments containing D-loops which marked the replication origin of chloroplast DNA were identified in two different species of Chlamydomonas. Each fragment was cloned in the E. coli plasmid pBR325. The cloned fragments were compared by restriction endonuclease analyses and by heteroduplex analyses in the electron microscope. The relative position of the D-loop regions and the homologous regions between the 2 fragments was determined. The D-loops were located within one short homologous region of 0. 42kb in length between the 2 cloned restriction fragments. The homologous region was subcloned in pBR322. Closed circular plasmid DNAs containing the short homologous region showed preferred denaturation in the D-loop region under physiological salt concentration which suggested that D-loop region was AT rich. Sequence divergence was detected at both ends of the D-loop region. Southern blot analyses indicated the presence of species-specific repetitive sequences within the divergent regions.

Base Sequence↗
PubMed1997

Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subtilis.

To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.

Bacillus subtilis↗
PubMed1997

Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis.

Neutral/alkaline two-dimensional gel electrophoresis is a robust, easily interpretable, sensitive technique that has yielded insights about the in vivo replication of many types of DNA, from multicopy yeast plasmids to single-copy chromosome regions in unsynchronized mammalian cells. It can provide information about directions of replication fork movement and locations of replication origins, termini, and pause sites. Especially when combined with its partner technique, neutral/neutral two-dimensional gel electrophoresis, it is a method of choice for investigation of unknown situations.

Animals↗
PubMed1999

Characterization of the functional replication origin of Mycobacterium tuberculosis.

The gene order in the 5kb Mycobacterium tuberculosis dnaA region is rnpA, rpmH, dnaA, dnaN and recF. We show that M. tuberculosis DNA fragment containing the dnaA-dnaN intergenic region functioned as oriC, i.e., allowed autonomous replication to otherwise nonreplicative plasmids, in M. tuberculosis H37Ra (H37Ra), avirulent strain of M. tuberculosis, and in Mycobacterium bovis BCG (BCG), a closely related, slowly growing mycobacterial strain. Removal of Escherichia coli plasmid replication origin (ColE1) from the M. tuberculosis oriC plasmids did not abolish their ability to function as oriC, confirming that the autonomous replication activity of these plasmids is due to the presence of the DNA fragment containing the dnaA-dnaN intergenic region. Deletion analyses revealed that the minimal oriC DNA fragment is 814bp. The copy number of M. tuberculosis oriC plasmids containing ColE1 ori relative to chromosomal oriC is one and the 5' flanking region of minimal oriC contains features that support stable autonomous replication. The M. tuberculosis oriC did not function in rapidly growing mycobacterial species such as M. smegmatis. M. smegmatis oriC functioned only in M. fortuitum, but not in any of the slowly growing mycobacterial species such as M. tuberculosis and BCG. Together these data suggest that the replication initiation mechanisms in the slowly growing Mycobacteria are similar and probably different from those in the rapidly growing Mycobacteria and vice versa.

Bacterial Proteins↗
PubMed1992

Opposed actions of regulatory proteins, DnaA and IciA, in opening the replication origin of Escherichia coli.

The opening of the three tandem 13-mers (iterons) in the replication origin (oriC) of Escherichia coli by DnaA protein, assisted by protein HU or IHF (Hwang, D. S., and Kornberg, A. (1992) J. Biol. Chem. 267, 23083-23086), represents an essential early stage in the initiation of chromosomal replication (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). We now show by mutational alterations of the 13-mer region that oriC function, both in vitro and in vivo, requires AT-richness in the left 13-mer and sequence specificity in the middle and right 13-mers. Interactions of DnaA protein with the middle and right 13-mers are crucial for the opening of the region. Binding of the protein to the top strand of the 13-mers appeared to maintain single-strandedness in the bottom strand. IciA protein, the inhibitor of initiation, binds the three 13-mers and blocks the opening of the region. The degrees of inhibition by IciA protein of 13-mer opening and of oriC plasmid replication observed with mutant forms of the 13-mers could be correlated with the binding affinity of IciA protein. Whereas the binding of IciA protein to the 13-mers did not affect the binding of DnaA protein to its four 9-mers boxes, interaction of DnaA protein with the 13-mers was blocked. The selective interactions of DnaA and IciA proteins with the 13-mer region appear to be components of the on/off switch that controls initiation of E. coli chromosomal replication.

Bacterial Proteins↗
PubMed1986

Interaction between gene II protein and the DNA replication origin of bacteriophage f1.

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.

Bacteriophages↗
PubMed1998

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921.

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.

Amino Acid Sequence↗
PubMed1996

An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp. strain PCC 7942.

In eubacteria, the clustering of DnaA boxes around the dnaN (beta subunit of DNA polymerase III) and dnaA genes usually defines the chromosome replication origin (oriC). In this study, the dnaN locus from the cyanobacterium Synechococcus sp. strain PCC 7942 was sequenced. The gene order in this region is cbbZp-dnaN-orf288-purL-purF which contrasts with other eubacteria. A cluster of eleven DnaA boxes (consensus sequence: TTTTCCACA) was found in the intergenic region between dnaN and cbbZp. We also found a 41-bp sequence within this region that is 80% identical to the proposed oriC of Streptomyces coelicolor. Therefore, we propose that this intergenic region may serve as an oriC in Synechococcus. Using bacterial luciferase as a reporter, we also showed that dnaN is rhythmically expressed, suggesting that DNA replication could be under circadian control in this organism.

Bacterial Proteins↗
PubMed1992

A position effect on the time of replication origin activation in yeast.

The chromosomes of eukaryotes are characterized by the mosaic nature of their replication--large regions of DNA that replicate early in S phase are interspersed with regions that replicate late. This pattern of early and late synthesis appears to be the consequence of a temporal program that activates replication origins at different times. The basis of this temporal regulation in the yeast S. cerevisiae has been investigated by changing the chromosomal locations of two origins, one activated early in the S phase (ARS1) and one activated late (ARS501). We show that the cis-acting information controlling time of activation can be separated from the element that determines origin function. For the ARS501 origin, late activation appears to be a consequence of its proximity to the telomere.

Chromosomes, Fungal↗
PubMed1994

Densely methylated DNA islands in mammalian chromosomal replication origins.

Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, oriS14, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-beta, is approximately 17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oriS14 is 127 bp long, and the DMI in ori-beta is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMI-mediated regulation of mammalian chromosomal origins are considered.

Animals↗
PubMed1993

Evidence of two levels of control of P1 oriR and host oriC replication origins by DNA adenine methylation.

A mutant mini-P1 plasmid with increased copy number can be established in Dam- strains of Escherichia coli, where mini-P1 plasmid replication is normally blocked. Comparison of this plasmid and a plasmid driven by the host oriC replication origin showed that both origins are subject to control by methylation at two different levels. First, both origins appear to be subject to negative regulation acting at the level of hemimethylation. This probably involves the sequestration of the hemimethylated DNA produced by replication, as has been previously described for oriC. Second, both origins show a positive requirement for adenine methylation for efficient function in vivo. This conclusion is supported by the behavior of the P1 origin in an improved in vitro replication system. In vitro, where sequestration of hemimethylated DNA is not expected to occur, the hemimethylated P1 origin DNA was fully functional as a template. However, the activity of fully unmethylated DNA was severely restricted in comparison with that of either of the methylated forms. This in vitro uncoupling of the two effects of origin methylation suggests that two separate mechanisms are involved.

Adenine↗
PubMed1995

Lymantria dispar nuclear polyhedrosis virus homologous regions: characterization of their ability to function as replication origins.

Homologous regions (hrs) were identified in the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) genome. A 1.58-kb region surrounding hr4 was sequenced and found to have two distinct domains. Domain I (about 600 bp) is composed of seven repeats of about 80 bp including a series of palindromes containing MluI sites and overlapping XhoI and SacI sites. Domain II (about 700 bp) is composed of eight partially repeated sequences of 60 to 100 bp containing a 15- to 25-bp sequence that is 80 to 100% A+T in addition to a 6- to 10-bp palindrome containing an NruI site. Hybridization of a domain I sequence to cosmids containing the LdMNPV genome indicated its presence at eight positions (hr1 to -8) on the genome. In contrast, hybridization of domain II indicated that it was present only at the hr4 locus. A DpnI-based transient-replication assay was used to determine if subclones of hr4 transfected into LdMNPV-infected L. dispar cells functioned as replication origins. Subclones of hr4 containing either domain I or domain II replicated at very low or moderate levels, respectively. However, when domain I and domain II were linked on the same plasmid, high levels of replication were observed. A 1.4-kb region containing hr1 was also sequenced. It lies immediately upstream of the polyhedrin gene and contains six domain I-type repeats. Four-hundred-base-pair regions of domain I repeats from hr1 and hr4 showed 89% sequence identity. Plasmids containing the hr1 domain I replicated at low levels. However, hybrid plasmids in which the AT-rich hr4 domain II was inserted adjacent to hr1 domain I replicated to high levels, indicating that the AT-rich domain II greatly enhances replication. The orientation and position of domains I and II relative to each other did not have major effects on the levels of replication.

Animals↗
PubMed1988

Studies on the role of the phi X174 gene A protein in phi X174 viral strand synthesis. III. Replication of DNA containing two viral replication origins.

Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins. Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination). In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin. When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins. Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination. In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products. This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave. Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined. With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence. Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred.

Bacteriophage phi X 174↗
PubMed2003

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin.

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.

Amino Acid Sequence↗
PubMed1984

Sequence organization of replication origin of the Escherichia coli K-12 chromosome.

A sequence of 245 base-pairs (oriC) in the replication origin of the Escherichia coli K-12 chromosome has been shown to provide all the information essential for initiation of bidirectional replication. In order to elucidate the sequence organization of oriC, numerous mutants carrying a single-to-multiple transitions from G X C to A X T base-pair were constructed by localized mutagenesis in vitro, which uses sodium bisulfite, and the correlation between the mutation sites and replicating ability (Ori function) was systematically analyzed. By isolating non-defective (Ori+) mutants with multiple base changes, transitions at 71 positions among 101 G X C pairs in oriC were found to have no effect on Ori function. Investigation of defective (Ori-) mutants, on the other hand, showed that individual replacements at 18 positions were detrimental to Ori function to some extent. These irreplaceable G X C pairs fell in the positions where no substitution was detected in the Ori+ mutants. The defect of the Ori- mutants with a single base substitution was generally weaker than that of the previously constructed Ori- mutants lacking a part of oriC. The addition of two or more base changes each giving a faint Ori- phenotype, however, resulted in a more intensive Ori- phenotype. We have previously demonstrated that oriC contains several regions where deletion or insertion of oligonucleotides leads to strong Ori- phenotypes. Transitions in those areas did not cause any defect of Ori function. Combining present results on base substitution mutants with the previous observations together, we assumed that the oriC sequence provides multiple interaction sites with replication initiation factors, and the precise arrangement of these sites are required for Ori function.

Base Composition↗