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At least 667 records · Page 37Linked to original sources
PubMed1992

A new chick mitochondrial DNA-binding protein exhibits sequence-specific interaction near heavy-strand replication origin: cleavage activity, stimulation of mtDNA synthesis, and enhancement in transformed fibroblasts.

We have identified a new, double-strand-dependent, mtDNA-binding protein in chick embryo fibroblast (CEF) mitochondria (and inner-membrane-matrix preparations) which demonstrates both an exclusive specific affinity for the displacement loop (D-loop) control region of chick mtDNA and intramitochondrial levels that reflect corresponding changes in mtDNA replication activity both in vivo and in vitro. This approximately 36 kDa protein (designated aMDP1, avian mitochondrial DNA-binding protein 1) was identified by elution and renaturation following SDS-polyacrylamide gel electrophoresis and by direct isolation from specific mtDNA-protein complexes excised from mobility shift gels. Analysis of the entire 16.7-kb mt genome determined that a MDP1 mediates cleavage of chick mtDNA in vitro at three H- and two L-strand sequence-specific target sites located within a 90-bp A + T-rich genomic tract, theoretically capable of forming stable secondary structures, approximately 200 bases upstream from the H-strand origin (OH) of replication. Furthermore, gel-isolated aMDP1 relaxes supercoiled mtDNA, and exogenous addition of the protein, in a permeabilized in vitro system, preferentially stimulates the synthesis of H-strand sequences which hybridize to OH-containing fragments. Oncogenic transformation of CEF by Rous sarcoma viruses results in a threefold elevated level of aMDP1, directly correlating with a similarly increased level of mtDNA replication in vivo. Heterologous chick-human cross-competition experiments showed that aMDP1 also selectively interacts with human (HeLa) D-loop region mtDNA, possibly reflective of an evolutionary importance for aMDP1 interaction in the region. Functionally, we hypothesize that aMDP1 may operate in conjunction with other mtDNA-binding proteins, important in replication and transcription, by potentiating duplex unwinding either prior to or during an initial stage of H-strand synthesis. Together, these results suggest that aMDP1 is a good potential candidate for a nucleus-encoded regulatory protein which communicates with the mt genome during the replication process.

Animals↗
PubMed2001

A potential role for mini-chromosome maintenance (MCM) proteins in initiation at the dihydrofolate reductase replication origin.

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.

Animals↗
PubMed1992

New IS10 transposition vectors based on a gram-positive replication origin.

We describe below a set of plasmid-based vehicles which can be used for delivery of IS10-derived transposons into Gram- bacteria. These vehicles replicate via a Gram+ plasmid origin that is inactive in Escherichia coli; they are easily maintained in Bacillus subtilis. Transposons are introduced by electroporation or transformation with the plasmid, and as in previous delivery systems, transpositions are selected with the appropriate antibiotic. This system should be particularly useful in situations where the standard delivery vehicles, based on bacteriophage lambda, are inappropriate. The system described incorporates a number of useful features: a variety of antibiotic markers (Er, Cm, Km or Tc), a polylinker containing restriction sites for rare-cutting endonucleases to facilitate physical mapping of chromosomal insertions, a mutant transposase that confers a relaxation in insertion specificity and positioning of the transposase-encoding gene outside of the transposing segment to ensure the stability of insertions once isolated.

Bacillus subtilis↗
PubMed1987

Protein-DNA interaction within one cloned chloroplast DNA replication origin of Chlamydomonas.

A partially purified algal protein mixture which supports in vitro DNA replication consists of soluble proteins and proteins extracted from thylakoid membrane. The membrane extract is essential for the specific initiation of replication at a displacement loop (D-loop) site previously mapped by electron microscopy. D-loop site and its flanking sequences have been cloned and sequenced. In this study, fragment-retention assays using various subclones of the sequenced region indicate that some proteins in the membrane extract bind strongly and specifically with a 494 bp restriction fragment which partially overlaps the D-loop site. Protein gel analyses of the protein-DNA complex identify three DNA-binding polypeptides with apparent molecular weights of 18, 24 and 26 kDA, respectively. Treatment with chloramphenicol, an inhibitor of chloroplast protein synthesis, for 1 h has no obvious effect on the contents of the 24 or 26 kDa polypeptides but significantly reduces the content of the 18 kDa polypeptide in the membrane extract.

Animals↗
PubMed1986

Prokaryotic promoters in the chloroplast DNA replication origin of Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, one displacement loop region which initiates the replication of chloroplast DNA was located on a 1.05 kb restriction fragment. This fragment was cloned and sequenced. In this report, the galK expression plasmid, pKO1 was used to screen for the presence of any prokaryotic promoter within the cloned fragment. The insertion of 2 AluI fragments yielded galK+ colonies. Sequence analyses of these AluI inserts revealed prokaryotic promoter consensus regions. Cloning into pKOTWI and subsequent DNA sequencing were used to determine the promoter-active orientation of each insert. Two back-to-back prokaryotic promoters were mapped on a 79 bp AluI fragment located within the displacement loop region.

Chlamydomonas↗
PubMed1995

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein.

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.

Amino Acid Sequence↗
PubMed1983

Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid.

A primosome assembly site for F plasmid DNA replication has been identified. This site, which we term rriA (F), is localized to one strand of a 385-base-pair Sau3A restriction fragment very close to ori 2 and within the 2.25-kilobase DNA sequence required for replication and incompatibility of the entire F plasmid. rriA (F) was isolated by cloning into the deletion phage vector M13 delta Elac. This phage forms very faint plaques due to a deletion of the M13 complementary strand origin but forms large wild-type plaques when DNA single-strand initiation determinants are inserted. The single-stranded viral DNA of the Sau3A F-M13 delta Elac recombinant provides an effector site of dATP hydrolysis by the primosomal protein n'. It also provides an assembly site for the Escherichia coli primosome protein complex that directs the in vitro conversion of the single-stranded DNA to a double-stranded form by the same mechanism as that used by phi X174. Homologies of the nucleotide sequence between this F DNA sequence and the previously identified primosome assembly sites in phi X174 phage DNA and in ColE1 plasmid DNA (rriA and rriB) have been found. The sequences 5' G-T-G-A-G-C-G 3' and 5' G-N-G-G-A-A-G-C 3' or variations of these sequences occur from two to five times within each assembly locus. In addition, two distinct 15-base-pair sequences in rriA (F) are perfectly homologous to corresponding sequences in rriA (ColE1).

Bacterial Proteins↗
PubMed1992

The replication origin of the Methylomonas clara plasmid pBE-2.

The Methylomonas clara narrow host range plasmids pBE-2 and pBE-3 belong to the class of plasmids encoding a trans acting replication initiation factor. Characteristically for such plasmids, the sequence of the origin of pBE-2 and pBE-3 contains a number of large direct repeats (8 and a half iterons of 19 bp), which by analogy are putative binding sites of the trans acting replication factor. Several additional features typical for the majority of E. coli plasmids were found in the M. clara origin: These include sequences homologous to the E. coli DnaA-box, sequences resembling E. coli IHF binding-sites, an AT-rich region with short repeats (similar to those repeats of E. coli origins responsible for an initial DNA duplex opening), and an AT-rich bent DNA region containing inverted repeats which have homology to small repeated sequences found in several plasmid origins. In addition, in the M. clara plasmid origin, large potential hairpin structures are present and the sequence of one of these participates in site specific recombination.

Base Sequence↗
PubMed1992

Mapping of a Physarum chromosomal origin of replication tightly linked to a developmentally-regulated profilin gene.

We compared the pattern of replication of two cell-type specific profilin genes in one developmental stage of the slime mold Physarum polycephalum. Taking advantage of the natural synchrony of S-phase within the plasmodium, we established that the actively transcribed profilin P gene is tightly linked to a chromosomal replication origin and is replicated at the onset of S-phase. In contrast, the inactive profilin A gene is not associated with a replication origin and it is duplicated in mid S-phase. Mapping by two-dimensional gel electrophoresis defines a short DNA fragment in the proximal upstream region of the profilin P gene from which bidirectional replication is initiated. We further provide an estimate of the kinetics of elongation of the replicon and demonstrate that the 2 alleles of the profilin P gene are coordinately replicated. All these results were obtained on total DNA preparations extracted from untreated cells. They provide a strong evidence for site specific initiation of DNA replication in Physarum.

Animals↗
PubMed1991

Probing the activation of the replicative origin of broad host-range plasmid R1162 with Tus, the E.coli anti-helicase protein.

The E.coli Tus protein is an anti-helicase involved in the termination of chromosome replication. The binding site for this protein, ter, was cloned into derivatives of the broad host-range plasmid R1162. The ter site caused the orientation-specific termination of plasmid replication fork movement in cell extracts containing Tus. Plasmids were constructed so that two sites for initiation of R1162 replication flanked the iteron-containing domain of the origin. In these plasmids, the site next to the AT-rich region within the iteron-containing domain was more active. In addition, when ter was placed between the more active site and the iterons, initiation of replication from this site was specifically inhibited. The data support a model for entry of the essential, plasmid-encoded helicase at one side of the direct repeats, and for its movement primarily in one direction away from these repeats to activate the initiation sites for DNA replication.

Bacterial Proteins↗
PubMed1990

The AT richness and gid transcription determine the left border of the replication origin of the E. coli chromosome.

We have identified novel, cis-acting elements which enhance in vivo the replication activity of plasmids carrying the minimal oriC of Escherichia coli. These are (i) the AT rich sequence ('AT-cluster') which exists immediately left of the 13mer repeats and (ii) the gid transcriptional unit. The 'AT-cluster' was functionally replaced by an unrelated AT rich sequence. This was also the case for the left and middle 13mers; they were substituted by the AT rich fragment from mini-F plasmid. The left 13mer was replaced by the AT rich sequence which did not show the 'reduced helical stability' known as the important character of the 13mer region. In contrast to these results, the right 13mer sequence was strictly required. As to the effect of the transcription from the gid promoter, the minimal oriC was activated only when the transcription was directed away from the left side of it. mioC transcription proceeding toward the oriC had no effect on the activation. Mutations in the DnaA boxes were partially suppressed by gid transcription leaving oriC from the left side. From these results, we propose that the AT richness is a determinant to identify the left border of oriC. It is presumed that gid transcription introduces negative superhelicity at the AT rich region and facilitates DnaA dependent duplex opening.

Adenine↗
PubMed1985

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region.

Approximately 10,000 nucleotides were sequenced in the oriC region of the Bacillus subtilis chromosome. The first replicating DNA strands are hybridized with a SalI-EcoRI fragment (nucleotide #1206-2954) in one direction (left to right) and an EcoRI-PstI fragment (#2949-4233) in the other. Seven open reading frames (ORF) accompanied with Shine-Dalgarno (SD) sequences were identified. ORF638 and ORF821 were identified as gyrB and gyrA genes respectively based on genetic evidences and amino acid sequence data. Comparison of amino acid sequences revealed that ORF44, ORF446, ORF378 and ORF323 are homologous with rpmH, dnaA, dnaN and recF of Escherichia coli, respectively. Thus, the organization of the ORFs from ORF44 to ORF638 resembles the organization of genes in the rpmH-gyrB region of the E. coli chromosome. Two non-coding regions characteristic for oriC signals were found near the site of initiation of the first replicating DNA. They are composed of repeating sequences whose consensus sequence TTAT(C/A)CACA is identical to that of 4 repeating sequences in the oriC of E. coli.

Amino Acid Sequence↗
PubMed1990

Multiple replication origins are used during Drosophila chorion gene amplification.

DNA from Drosophila egg chambers undergoing chorion gene amplification was analyzed using the two-dimensional gel technique of Brewer and Fangman. At stage 10, 34% of DNA molecules from the maximally amplified region of the third chromosome chorion gene cluster contained replication forks or bubbles. These nonlinear forms were intermediates in the process of amplification; they were confined to follicle cells, and were found only within the replicating region during the time of amplification. Multiple origins gave rise to these intermediates, since three separate regions of the third chromosome chorion locus contained replication bubbles. However, initiation was nonrandom; the majority of initiations appeared to occur near the Bgl II site located between the s18 and s15 chorion genes. The P[S6.9] chorion transposon also contained abundant replication intermediates in follicle cells from a transformed line. Initiation within P[S6.9] occurred near two previously defined cis-regulatory elements, one near the same Bgl II site (in the AER-d region) and one near the ACE3 element.

Animals↗
PubMed2000

MSSP, a protein binding to an origin of replication in the c-myc gene, interacts with a catalytic subunit of DNA polymerase alpha and stimulates its polymerase activity.

MSSP has been identified as a protein that binds to both single- and double-stranded sequences of a putative DNA replication origin sequence in the human c-myc gene. MSSP possesses versatile functions, including stimulation of DNA replication, transcriptional regulation, apoptosis induction, and cell transformation coordinated by c-Myc. MSSP contains two RNP domains, RNP1-A and RNP1-B, both of which are necessary for all of the functions of MSSP. In this study, we found that MSSP binds to the N-terminal region of a catalytic subunit of a human DNA polymerase alpha via its RNP domains both in vitro and in human cells. Furthermore, MSSP was released from the putative DNA replication origin of the c-myc gene after it complexed with DNA polymerase alpha, and MSSP stimulated DNA polymerase activity in vitro.

Cell Line↗
PubMed1986

Isolation of an episomal yeast gene and replication origin as chromatin.

A multicopy yeast plasmid containing the TRP1 gene (coding for N-5'-phosphoribosylanthranilate isomerase) and ARS1 (autonomously replicating sequence 1) has been purified as chromatin. Electrophoretic analysis of nucleic acid and proteins and electron microscopy show that the plasmid chromatin is largely free of contaminants. Electron-microscopic and linking-number analyses indicate that the plasmid chromatin contains seven nucleosomes, as predicted by the indirect end-label analyses of Thoma, Bergman, and Simpson [J. Mol. Biol. (1984) 177, 715-733]. Indirect end label mapping of micrococcal nuclease cuts demonstrates that nucleosome positions and nuclease-sensitive regions are not altered by the purification. The plasmid chromatin behaves homogeneously with respect to its elution from nuclei, template activity, and intrinsic buoyant density. Taken together, these observations suggest that different copies of the TRP1ARS1 plasmid do not differ from each other grossly in chromatin structure. We discuss the potential for understanding eukaryotic gene regulation offered by the ability to isolate unique genes as chromatin.

Aldose-Ketose Isomerases↗