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PubMed1992

Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF.

Opening of the three tandem repeats of a 13-mer in the replication origin (oriC) of Escherichia coli is a prime event in the replication in vitro of minichromosomes (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). DnaA, the initiator protein, requires protein HU or IHF, along with a millimolar level of ATP and negative superhelical density in the plasmid to open this region. The extent of opening, as judged by cleavage by a single-strand-specific endonuclease (i.e. P1 nuclease), correlated closely with replication of the oriC plasmid. In an initial complex, preceding opening of the 13-mers, the footprint of DnaA protein bound by ATP covered its four 9-mer recognition sequences. The footprint of the nucleotide-free form of the protein, by contrast, was more extensive and thus, less specific.

Bacterial Proteins↗
PubMed2000

A DNA fragment of Desulfovibrio gigas genome containing replication origin related genes.

The nucleotide sequence of a 10,772 base pair (bp) region from Desulfovibrio gigas genome was determined. This sequence, which is adjacent to the region containing the coding units for the metalloproteins rubredoxin-oxygen oxidoreductase (ROO) and rubredoxin, includes the flavodoxin gene. Additionally, it also contains four open reading frames (ORFs) related to genes frequently found in replication origin regions of prokaryotes. These hypothetical encoded polypeptides are: the response regulator proteins (PhoP and PhoR) from the phosphate regulon, a DNA partitioning protein and an asparagine synthetase.

Amino Acid Sequence↗
PubMed2000

Two widely spaced initiator binding sites create an HMG1-dependent parvovirus rolling-hairpin replication origin.

Minute virus of mice (MVM) replicates via a linearized form of rolling-circle replication in which the viral nickase, NS1, initiates DNA synthesis by introducing a site-specific nick into either of two distinct origin sequences. In vitro nicking and replication assays with substrates that had deletions or mutations were used to explore the sequences and structural elements essential for activity of one of these origins, located in the right-end (5') viral telomere. This structure contains 248 nucleotides, most-favorably arranged as a simple hairpin with six unpaired bases. However, a pair of opposing NS1 binding sites, located near its outboard end, create a 33-bp palindrome that could potentially assume an alternate cruciform configuration and hence directly bind HMG1, the essential cofactor for this origin. The palindromic nature of this sequence, and thus its ability to fold into a cruciform, was dispensable for origin function, as was the NS1 binding site occupying the inboard arm of the palindrome. In contrast, the NS1 site in the outboard arm was essential for initiation, even though positioned 120 bp from the nick site. The specific sequence of the nick site and an additional NS1 binding site which directly orients NS1 over the initiation site were also essential and delimited the inboard border of the minimal right-end origin. DNase I and hydroxyl radical footprints defined sequences protected by NS1 and suggest that HMG1 allows the NS1 molecules positioned at each end of the origin to interact, creating a distortion characteristic of a double helical loop.

Animals↗
PubMed1987

Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14.

The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the pi protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepB of pUB110 throughout the protein molecule, but not with RepC of pT181, pi of R6K or protein RepH encoded by and initiating the replication of pC194.

Amino Acid Sequence↗
PubMed2006

The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo.

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin.

Amino Acid Sequence↗
PubMed2006

Linear molecules of tobacco ptDNA end at known replication origins and additional loci.

Higher plant plastid DNA (ptDNA) is generally described as a double-stranded circular molecule of the size of the monomer of the plastid genome. Also, the substrates and products of ptDNA replication are generally assumed to be circular molecules. Linear or partly linear ptDNA molecules were detected in our present study using pulsed-field gel electrophoresis and Southern blotting of ptDNA restricted with 'single cutter' restriction enzymes. These linear DNA molecules show discrete end points which were mapped using appropriate probes. One possible explanation of discrete ends would be that they represent origins of replication. Indeed, some of the mapped ends correlate well with the known origins of replication of tobacco plastids, i.e. both of the oriA sequences and--less pronouncedly--with the oriB elements. Other ends correspond to replication origins that were described for Oenothera hookeri, Zea mays, Glycine max and Chlamydomonas reinhardtii, respectively, while some of the mapped ends were not described previously and might therefore represent additional origins of replication.

Blotting, Southern↗
PubMed1990

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.

Base Sequence↗
PubMed1991

A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K.

Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.

Bacterial Proteins↗
PubMed1989

The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli.

After binding to its four 9-mer boxes in the 245-base pair Escherichia coli replication origin (oriC), dnaA protein effects the formation of an "open complex" in an adjacent region made up of three 13-mers (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). This open complex formation requires the ATP form of dnaA protein assisted by HU protein (Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50, 259-265). We now provide direct evidence that dnaA protein binds the 13-mers, sequences that bear no resemblance to the 9-mer box. The evidence is (i) displacement of dnaA protein from the open complex by oriC or by a synthetic oligonucleotide containing the 13-mers, but not by a mutant of oriC lacking the 13-mers; (ii) filter binding of the synthetic (13-mer) oligonucleotide by dnaA protein; and (iii) requirement for the ATP form of dnaA protein assisted by HU protein for temperature-dependent binding to the 13-mer region. Controlled proteolysis of dnaA protein results in a prompt loss of oriC binding; an NH2-terminal 30-kDa peptide contains the domain that binds ATP and phospholipids known to destabilize the tightly bound ATP.

Adenosine Diphosphate↗
PubMed2003

Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins.

Actin-like proteins MreB and Mbl are required for proper cell shape and for viability in B. subtilis and form dynamic helical filaments underneath the cell membrane. We have found that depletion of MreB and Mbl proteins leads to a rapid defect in chromosome segregation before a defect in cell shape becomes detectable. Under these conditions, the SMC chromosome segregation complex that is essential for proper chromosome arrangement and segregation loses its specific subcellular localization, and replication origins fail to localize in a regular bipolar manner as in wild type cells. Time-lapse microscopy showed that during depletion of MreB, origin regions can move towards the same cell pole, showing that bipolar orientation of origin separation is lost. Contrarily, depletion of three other cell shape determinants, MreC, MreD, or MreBH (the third B. subtilis actin homolog) had no effect on chromosome segregation but varying effects on cell morphology. Depletion of MreC and MreD resulted in formation of round cells, while depletion of MreBH led to formation of vibrio-shaped cells. The data show that actin proteins Mbl and MreB are required for proper chromosome segregation and that Mre proteins affect different aspects in cell shape.

Actins↗
PubMed1988

Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of asnC transcripts.

Transcription from the asnC promoter was found to proceed through the replication origin, oriC, into the gidA gene of Escherichia coli. Between 10% and 20% of asnC transcripts reached oriC in vivo. Termination sites in the intergenic region between asnC and mioC and within mioC were determined in vivo and in vitro using S1 mapping. Only about 10% of the transcripts terminated at the asnC terminator in vivo. DnaA protein dependent termination was observed close to the binding site, dnaA box, for DnaA protein. In the in vitro replication system asnC transcripts did not reach oriC, suggesting that asnC transcripts are not involved in the initiation of replication, contrary to mioC transcripts. We suggest that oriC and mioC might have been transposed during evolution into an asnC regulation.

Bacterial Proteins↗
PubMed2001

Both heavy strand replication origins are active in partially duplicated human mitochondrial DNAs.

The replication of human mitochondrial DNA (mtDNA) is initiated from a pair of displaced origins, one priming continuous synthesis of daughter-strand DNA from the heavy strand (OH) and the other priming continuous synthesis from the light strand (OL). In patients with sporadic large-scale rearrangements of mitochondrial DNA (i.e., partially-deleted [Delta-mtDNA] and partially-duplicated [dup-mtDNA] molecules), the dup-mtDNAs typically contain extra origins of replication, but it is unknown at present whether they are competent for initiation of replication. Using cybrids harboring each of two types of dup-mtDNAs-one containing two OHs and two OLs, and one containing two OHs and one OL-we used ligation-mediated polymerase chain reaction (LMPCR) to measure the presence and relative amounts of nascent heavy strands originating from each OH. We found that the nascent heavy strands originated almost equally from the two OHs in each cell line, indicating that the extra OH present on a partially duplicated mtDNA is competent for heavy strand synthesis. This extra OH could potentially confer a replicative advantage to dup-mtDNAs, as these molecules may have twice as many opportunities to initiate replication compared to wild-type (or partially deleted) molecules.

Cell Line↗
PubMed1991

Transcriptional enhancer in the vicinity of a replication origin within the 5' region of the chicken alpha-globin gene domain.

A transcriptional enhancer is located near a replication origin within an upstream area of chicken domain of alpha-globin genes. Minimal region possessing enhancer properties is located about 4 kb upstream from the alpha-pi gene. Addition of the neighbouring 500 base-pair DNA fragment, including the constitutive DNase I hypersensitive site, increases the enhancer activity approximately twofold.

Animals↗
PubMed1993

The pSC101 par locus alters protein-DNA interactions in vivo at the plasmid replication origin.

We report here direct evidence that mutations in the par locus affect protein-DNA interactions in vivo at the replication origin of plasmid pSC101. Concomitant with par-mediated plasmid stabilization, two sites in the origin region show an altered methylation pattern as detected by in vivo footprinting with dimethyl sulfate. One site is located near an integration host factor-binding sequence adjacent to the first of three direct repeats known to be involved in the initiation of pSC101 replication; the second site is within the third direct repeat.

Base Sequence↗
PubMed1984

Initiation signals for complementary strand DNA synthesis in the region of the replication origin of the Escherichia coli chromosome.

We have used an in vivo plasmid-phi X174 packaging system to detect replication initiation signals in the region of the replication origin (oriC) of the Escherichia coli chromosome. The results obtained are summarized as follows: (i) Neither within nor close to oriC effective signals for initiating complementary strand synthesis could be detected. We conclude that initiation mechanisms for leading and lagging strand synthesis at oriC are not identical to any known priming mechanism of DNA synthesis. (ii) At least five signals that can function as complementary strand origins on ss-plasmid DNA are located in a region about 2000-3300 base pairs away from oriC in the clockwise direction on the chromosome. We suggest that these signals are protein n' like recognition sequences since they are dependent for their activity on dnaB protein and show sequence similarities to other putative n' recognition sequences. Surprisingly, some of the signals are located on the template for leading strand synthesis.

Bacteriophage phi X 174↗
PubMed1996

CTG triplet repeats from the myotonic dystrophy gene are expanded in Escherichia coli distal to the replication origin as a single large event.

The expansion and contraction of CTG and CGG trinucleotide repeat sequences have been associated with several heritable genetic diseases. We developed a system for investigating the expansion of triplet repeat sequences in Escherichia coli in order to elucidate molecular mechanisms. Analysis of expanded regions using the interrupting CTA triplet sequence as a location marker within the CTG tract revealed that the expansion of large CTG repeats is one event rather than an accumulation of multiple small expansions and that the expansions occur more frequently in the region distal from the replication origin. Also, we showed that a loss of interruptions increases the expansion frequency. Thus, the instability of large triplet repeats in hereditary diseases occurs by a mechanism different from the instability in microsatellite sequences caused by defects in mismatch repair systems for certain sporadic cancers and hereditary non-polyposis colorectal cancers.

Base Sequence↗
PubMed1991

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.

Amino Acid Sequence↗
PubMed1998

Replication origin of the broad host range plasmid RK2. Positioning of various motifs is critical for initiation of replication.

The 393-base pair minimal origin, oriV, of plasmid RK2 contains three iterated motifs essential for initiation of replication: consensus sequences for binding the bacterial DnaA protein, DnaA boxes, which have recently been shown to bind the DnaA protein; 17-base pair direct repeats, iterons, which bind the plasmid encoded replication protein, TrfA; and A + T-rich repeated sequences, 13-mers, which serve as the initial site of helix destabilization. To investigate how the organization of the RK2 origin contributes to the mechanism of replication initiation, mutations were introduced into the minimal origin which altered the sequence and/or spacing of each particular region relative to the rest of the origin. These altered origins were analyzed for replication activity in vivo and in vitro, for localized strand opening and for DnaB helicase mediated unwinding. Mutations in the region between the iterons and the 13-mers which altered the helical phase or the intrinsic DNA curvature prevented strand opening of the origin and consequently abolished replication activity. Insertions of more or less than one helical turn between the DnaA boxes and the iterons also inactivated the replication origin. In these mutants, however, strand opening appeared normal but the levels of DnaB helicase activity were substantially reduced. These results demonstrate that correct helical phasing and intrinsic DNA curvature are critical for the formation of an open complex and that the DnaA boxes must be on the correct side of the helix to load DnaB helicase.

Bacterial Proteins↗