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PubMed2002

Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids.

Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10(-6) transformants/CFU/ micro g of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.

Bacterial Proteins↗
PubMed1988

Circular ribosomal DNA plasmids transform Tetrahymena thermophila by homologous recombination with endogenous macronuclear ribosomal DNA.

We transformed the ciliate Tetrahymena thermophila by microinjection of circular plasmids containing the ribosomal RNA gene (rDNA). In the somatic macronucleus of Tetrahymena, the rDNA is in the form of linear palindromic molecules. The rDNA molecules from the C3 strain have a replication advantage over rDNA from both B strain and the C3 rDNA mutant rmm1. We constructed two circular plasmids carrying replication origin sequences from C3 rDNA and a point mutation (Pmr) in the 17S rRNA gene that confers resistance to the antibiotic paromomycin. One plasmid contained a single complete copy of the rRNA gene and its flanking sequences, while the other had an additional rDNA origin of replication. In all B or rmm1 Tetrahymena cell lines transformed with the plasmids, rDNA sequences from the plasmid were found in palindromic rDNA molecules. In one transformant line, a small amount of the plasmid was also retained in a form with the original circular restriction map. Our results show that the plasmids underwent homologous recombination with one arm of the endogenous rDNA to give heteropalindromic rDNA, or with both arms of the palindrome to form homopalindromic rDNA. The resulting recombinant molecules were able to replace the recipient's original rDNA completely, providing strong evidence that C3 rDNA sequences in the donor DNAs confer a replication advantage over recipient rDNA. Thus microinjection of circular plasmids provides a method for replacement of an endogenous gene or gene fragment with exogenous sequences.

Animals↗
PubMed2005

A study on the correlation of nucleotide skews and the positioning of the origin of replication: different modes of replication in bacterial species.

Deviations from Chargaff's 2nd parity rule, according to which A approximately T and G approximately C in single stranded DNA, have been associated with replication as well as with transcription in prokaryotes. Based on observations regarding mainly the transcription-replication co-linearity in a large number of prokaryotic species, we formulate the hypothesis that the replication procedure may follow different modes between genomes throughout which the skews clearly follow different patterns. We draw the conclusion that multiple functional sites of origin of replication may exist in the genomes of most archaea and in some exceptional cases of eubacteria, while in the majority of eubacteria, replication occurs through a single fixed origin.

Archaea↗
PubMed2001

Replication licensing of the EBV oriP minichromosome.

The latent EBV genome may persist in the integrated form as well as the circular episomal form. However, most of the latent viral DNA molecules are known to exist in the circular episomal form, which binds to host chromosomes during mitosis. The DS element of oriP in the circular episomal DNA functions as a replication origin. As it replicates once in a single S phase, it is possible that oriP is regulated by the cellular replication licensing mechanism including the MCM family of replication licensing factors. Transient replication analysis using the oriP plasmid and HeLa/EB1 cells revealed that the DS element requires early G1 phase for the next round of replication, the same cell-cycle window in which the replication licensing of cellular chromatin occurs. After this phase, the sedimentation velocity of the oriP minichromosome increases. MCM2 associates with the oriP minichromosome at late G1 but not at G2/M, and this association requires the DS element in the plasmid. The interaction of EBNA1 and the MCM proteins on the DS element was also suggested. These results suggested that the cellular licensing mechanism controls the replication from oriP. This also suggested a similarity in the replication machinery of the cellular chromatin and the latent EBV genome. In addition to DS-dependent replication, the EBV genome replicates in a manner independent of the DS element in several cultured cell lines. The DS-dependent replication is likely to be suppressed in these cell lines by the expression of other viral proteins. In contrast, EBV-positive Burkitt's lymphoma and circulating EBV-infected B cells express only EBNA1 or both EBNA1 and LMP2. DS-dependent replication may play a major role in these EBNA1-only cells, and the licensing regulation of oriP is important for maintenance of the EBV genome during this latent period of the viral life cycle. EBNA1 is required for efficient nuclear retention and partitioning of oriP-carrying plasmid by its binding to the FR element, thus providing stable persistence of the latent EBV genome during cell division. The copy number of latent EBV DNA molecules in B-cell lines remains fairly constant during multiple passage in culture. However, very little is known about the mechanism by which the viral DNA molecules are equally segregated into daughter cells. To understand the mechanisms responsible for stable nuclear retention and partitioning of the latent viral genome, it is essential to analyze the episomal and integrated viral DNAs at a single-cell level by FISH and other techniques.

Cell Cycle↗
PubMed1993

Characterization of factors that suppress linear DNA replication in SV40 in vitro replication system.

The in vitro simian virus 40 (SV40) replication system has been developed as a model system of cellular DNA replication, because the replication initiated from the replication origin of SV40 and replication fork proceeds bidirectionally. In this system, SV40 T-antigen (TAg) is the only factor provided by viral genes, while all other factors are supplied by the host cells. A suppression of replication has been observed in the linear template containing SV40 replication origin, compared with the closed circular template in the SV40 in vitro replication system using a crude extract of HeLa cells. However in the in vitro replication system reconstituted from partially purified factors, less preference was observed for the replication of the closed circular DNA over the linear DNA. In a mono-polymerase system supplemented by crude extracts, a suppression of replication in a linear template was also observed, when compared with a closed circular template. This suppression effect of crude extract was abolished by heat treatment, suggesting that the suppression was induced by some protein factors. A crude extract of HeLa cells was fractionated by stepwise elution with buffers containing 0.2 M, 0.4 M, 0.6 M and 1 M NaCl on a phosphocellulose column, and characterization of factors that suppress linear DNA replication has been done. Both fractions that were eluted at 0.4 M and 0.6 M from phosphocellulose were necessary to suppress linear DNA replication efficiently. The factors in the 0.6 M fraction that suppressed linear DNA replication synergistically with the 0.4 M fraction were partially purified by successive chromatography with heparin-sepharose and dsDNA-cellulose followed by glycerol gradient centrifugation. These results suggested that multiple factors are required to suppress DNA replication of the linear template.

Cell Fractionation↗
PubMed1996

Characterization of a binding site for the herpes simplex virus type 1 UL9 origin-binding protein within the UL9 gene.

Gene UL9 of herpes simplex virus type 1 (HSV-1) encodes a sequence-specific origin-binding protein (OBP) that plays a direct and essential role in viral DNA synthesis. A search of the complete HSV-1 genomic sequence for possible OBP binding sites lying outside the known origins of replication revealed the presence of a very close match to the OBP recognition sequence within the UL9 coding region. The ability of OBP to bind to this site (referred to as the 'UL9 box') was confirmed by DNase I footprinting and gel retardation assays, and filter binding experiments demonstrated that the affinity of OBP for the UL9 box was of the same order as for its high affinity sites within the three replication origins. To investigate whether binding of OBP to the UL9 box played a role during viral replication we constructed a mutant virus in which the sequence was altered in such a way as to preserve the encoded amino acid sequence whilst abolishing the ability of OBP to bind. Growth of the virus was indistinguishable from wild-type and no alterations were observed in the accumulation of transcripts from the UL9 region of the genome. In addition, a DNA fragment containing the UL9 box sequence did not exhibit origin activity in a transient assay for viral DNA synthesis. We therefore conclude that binding of OBP to the UL9 box is not essential for virus growth and that expression of the UL9 gene is unlikely to be autoregulated through this site.

Base Sequence↗
PubMed1995

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01.

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 bp region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR I) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage phi X174. This IR was shown to have a partial, RNA polymerase-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerase-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).

Bacteriophage M13↗
PubMed1997

Identification of HsORC4, a member of the human origin of replication recognition complex.

A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. HsORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex.

Amino Acid Sequence↗
PubMed1990

Deletion analysis of the mini-P1 plasmid origin of replication and the role of Escherichia coli DnaA protein.

The mini-P1 plasmid origin of replication is contained on a 246 base pair (bp) piece of DNA. At one end there are five 19-bp binding sites for the P1 initiator protein, RepA, and near the other end there are two 9-bp DnaA protein-binding sites. To further define the limits of the origin, we cloned the origin region in M13 and constructed deletions of either end. We sequenced the DNA and tested the replicative form I DNA of the deletion phages for their ability to support RepA-dependent DNA replication in an in vitro system. The origin that is functional in vitro could be reduced to 202 bp. It includes three intact and one incomplete RepA-binding sites at one end and the two DnaA-binding sites at the other end. When the two naturally occurring DnaA-binding sites were replaced with one or two synthetic sites, only the construction containing two sites was active in vitro. We found that the minimal origin that is functional in vivo contains all of the five RepA and the two DnaA-binding sites. Mini-P1 plasmid replication both in vivo and in vitro requires two initiator proteins, the Escherichia coli DnaA protein and the P1 RepA protein. We have found that the ADP form of DnaA is as active as the ATP form of the protein in the in vitro replication of mini-P1. In contrast, only the ATP form is active for in vitro replication of plasmids carrying the E. coli origin (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755).

Adenosine Diphosphate↗
PubMed2000

DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair.

DNA damage formation and repair are tightly linked to protein-DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication ( ARS1 ). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct inter-actions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.

DNA Damage↗
PubMed2006

Tolerance of Sir1p/origin recognition complex-dependent silencing for enhanced origin firing at HMRa.

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.

Binding Sites↗
PubMed1992

EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication.

The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-1p cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNA1 does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNA1 does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNA1. This retention is specific for DNA fragments containing EBNA1-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.

Adenosine Triphosphatases↗
PubMed1991

Single-stranded structures are present within plasmids containing the Epstein-Barr virus latent origin of replication.

The Epstein-Barr virus (EBV) latent origin of plasmid replication (oriP) contains two essential regions, a family of repeats with 20 imperfect copies of a 30-bp sequence and a dyad symmetry element with four similar 30-bp repeats. Each of the repeats has an internal palindromic sequence and can bind EBNA 1, a protein that together with oriP constitutes the only viral element necessary for EBV maintenance and replication. Using single-strand-specific nucleases, we have probed plasmids containing oriP-derived sequences for the presence of secondary structural elements. Multiple single-stranded structures were detected within the oriP region. Of the two essential elements of oriP, the family of repeats seemed to extrude these structures at a much higher frequency than did sequences within the dyad symmetry region. Though negative supercoiling was found to stabilize the single-stranded structures, they showed significant stability even after linearization of the oriP plasmids. Two major single-stranded structures detected involved approximately 12 bp of DNA. These loci could be transiently unwound regions that form because of negative supercoiling and the high A + T content of this region of DNA, or they could be cruciform structures extruded within the palindromic sequences of oriP that may be important sites for protein-DNA interactions in the EBV oriP.

Antigens, Viral↗
PubMed2001

Rep and Rep' protein of porcine circovirus type 1 bind to the origin of replication in vitro.

Genome replication of Porcine circovirus type 1 (PCV1) relies upon expression of the full-length protein Rep and a spliced isoform (Rep'), and the presence of a 111-bp genomic fragment comprising the origin of replication. Using an electrophoretic mobility shift assay (EMSA), the capability of both Rep proteins to bind to partial fragments of the origin of replication of PCV1 was investigated in vitro. Both proteins formed complexes with double-stranded DNA origin fragments containing a stem-loop structure with a conserved nonamer and four hexamer repeats (5'-CGGCAG; H1 to H4). Use of truncated EMSA substrates identified minimal binding sites (MBS) for Rep and Rep' protein: The Rep binding site was mapped to the right leg of the stem-loop and the two inner hexamer repeats H1/H2, while binding of Rep' required only the presence of two hexamer repeats. Two differentially retarded complexes were observed with Rep protein, which presumably result from alternative binding to the MBS or to H3/4.

Animals↗
PubMed1989

Topoisomerase I sites cluster asymmetrically at the ends of the simian virus 40 core origin of replication.

In vivo, topoisomerase I cleavage sites are located predominantly on the strands of simian virus 40 DNA that are the templates for discontinuous synthesis (S.E. Porter and J.J. Champoux, Mol. Cell. Biol. 9:541-550, 1989). This arrangement of sites suggests that topoisomerase I may associate with replication complexes in unique functional orientations at replication forks. We have mapped topoisomerase I cleavage sites in the simian virus 40 origin of replication in vitro under conditions suitable for DNA replication. Numerous sites cluster in the inverted repeat and AT-rich domains at the ends of the core origin and are arranged on the same strands that are cut most frequently in vivo. We propose that cleavage at these sites would allow bidirectional extension of the replication bubble induced by T antigen within the core origin of replication early in the initiation of DNA synthesis. A mutational analysis of the topoisomerase I sites confirms the importance of positions -4 to -1 and +1 in the consensus sequence 5'-A/T-A/G-A/T-T-break-G/A-3'. Surprisingly, more distant nucleotide positions also influence topoisomerase I sites in the inverted repeat and AT-rich domains of the core origin. The effects of distant sequences could be mediated by direct interactions with topoisomerase I or by the conformation of DNA in the core origin.

Animals↗
PubMed2001

DNA sequence heterogeneity within the Epstein-Barr virus family of repeats in the latent origin of replication.

To detect the presence of variability in the tandemly repeated sequences of the Epstein-Barr virus latent origin of replication, we analyzed the length of the family of repeats in 14 lymphoblastoid and Burkitt's lymphoma cell lines by PCR amplification. The gel electrophoresis analysis of the PCR products revealed a broad banding pattern, characteristic of each line, consisting of several fragments, sometimes smeared, of variable length. This finding was interpreted as a result of the hairpin-like structures generated by the palindrome within the family of repeats, able to originate artefacts. Since the banding pattern was different only in strictly non-correlated cell lines, we supposed that the sequence of the repeat units was polymorphic. We therefore sequenced the family of repeats in three healthy bone marrow derived lymphoblastoid cell lines carrying an endogenous EBV as well as in a B95-8 infected cell line as control. The sequence analysis revealed that each line is different both in the number and in the sequence of repeats. At the 3' end of the family of repeats the B95-8 virus was found to have a 252 bp region missing in the GenBank standard sequence. This one is probably a partial sequence since it was shorter than the control specimens obtained from different sources of B95-8 DNA analyzed by Southern blot hybridization. The length analysis of the family of repeats can be used to characterize EBV strains by PCR.

Base Sequence↗
PubMed1996

Cooperative assembly of EBNA1 on the Epstein-Barr virus latent origin of replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates DNA replication by binding to multiple copies of its 18-bp recognition sequence present in the Epstein-Barr virus latent origin of DNA replication, oriP. Using electrophoretic mobility shift assays, we have localized the minimal DNA binding domain of EBNA1 to between amino acids 470 and 607. We have also demonstrated that EBNA1 assembles cooperatively on the dyad symmetry subelement of oriP and that this cooperative interaction is mediated by residues within the minimal DNA binding and dimerization domain of EBNA1.

Antigens, Viral↗
PubMed1993

Bacterial chromosome origins of replication.

Bacteria regulate chromosomal replication from one specific origin. We compare the regulatory requirements, DNA structures, and biochemical properties of the prototypic Escherichia coli origin with those of evolutionarily distant Bacillus subtilis and Caulobacter crescentus origins. The ubiquitous DnaA protein is a major regulator of all three bacterial origins. Unique features of these origins, however, may reflect specific regulatory requirements placed on them.

Bacteria↗