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PubMed1986

The DNA replication origins of herpes simplex virus type 1 strain Angelotti.

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.

Animals↗
PubMed2002

Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus.

How eukaryotes specify their replication origins is an important unanswered question. Here, we analyze the replicative organization of yeast rDNA, which consists of approximately 150 identical repeats, each containing a potential origin. Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced. This repression is largely mediated by the Sir2p histone-deacetylase. Increased origin firing in sir2 Delta mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging. We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination.

DNA Replication↗
PubMed2005

Replication-associated strand asymmetries in mammalian genomes: toward detection of replication origins.

In the course of evolution, mutations do not affect both strands of genomic DNA equally. This imbalance mainly results from asymmetric DNA mutation and repair processes associated with replication and transcription. In prokaryotes, prevalence of G over C and T over A is frequently observed in the leading strand. The sign of the resulting TA and GC skews changes abruptly when crossing replication-origin and termination sites, producing characteristic step-like transitions. In mammals, transcription-coupled skews have been detected, but so far, no bias has been associated with replication. Here, analysis of intergenic and transcribed regions flanking experimentally identified human replication origins and the corresponding mouse and dog homologous regions demonstrates the existence of compositional strand asymmetries associated with replication. Multiscale analysis of human genome skew profiles reveals numerous transitions that allow us to identify a set of 1,000 putative replication initiation zones. Around these putative origins, the skew profile displays a characteristic jagged pattern also observed in mouse and dog genomes. We therefore propose that in mammalian cells, replication termination sites are randomly distributed between adjacent origins. Taken together, these analyses constitute a step toward genome-wide studies of replication mechanisms.

Animals↗
PubMed1983

Maintenance and incompatibility of plasmids carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication between oriC and asnA.

Plasmids that replicate only by means of the cloned Escherichia coli replication origin (oriC) are called minichromosomes or oriC-plasmids. In this paper it is shown that sequences located between oriC and asnA are involved in maintenance and incompatibility of minichromosomes. These sequences include part of the 16kD and 17kD genes, previously allocated within this region (1,2). Transcription towards oriC that is initiated at the 16kD promoter, specifically enhances the stability and copy-number of minichromosomes. Three regions are involved in minichromosome incompatibility. One region, incA, includes the minimal oriC sequence. A second, incB, maps within a 210 base pairs fragment that overlaps the 16kD promoter. The third, incC, encompasses the 17kD gene. Neither one of the regions expresses incompatibility on its own, but the additional presence of one of the others is required. The data presented indicate that sequences of the 16kD and 17kD genes are part of the replication control system of oriC-plasmids.

Base Sequence↗
PubMed2004

Redox potential regulates binding of universal minicircle sequence binding protein at the kinetoplast DNA replication origin.

Kinetoplast DNA, the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a remarkable structure containing 5,000 topologically linked DNA minicircles. Their replication is initiated at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L- and H-strands, respectively. A UMS-binding protein (UMSBP), binds specifically the conserved origin sequences in their single stranded conformation. The five CCHC-type zinc knuckle motifs, predicted in UMSBP, fold into zinc-dependent structures capable of binding a single-stranded nucleic acid ligand. Zinc knuckles that are involved in the binding of DNA differ from those mediating protein-protein interactions that lead to the dimerization of UMSBP. Both UMSBP DNA binding and its dimerization are sensitive to redox potential. Oxidation of UMSBP results in the protein dimerization, mediated through its N-terminal domain, with a concomitant inhibition of its DNA-binding activity. UMSBP reduction yields monomers that are active in the binding of DNA through the protein C-terminal region. C. fasciculata trypanothione-dependent tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. The possibility that UMSBP binding at the minicircle replication origin is regulated in vivo by a redox potential-based mechanism is discussed.

Animals↗
PubMed1994

Parental strand recognition of the DNA replication origin by the outer membrane in Escherichia coli.

The outer membrane of Escherichia coli binds the origin of DNA replication (oriC) only when it is hemimethylated. We report here the results of a footprinting analysis with the outer membrane which demonstrate that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated. Two regions, flanking the Integration Host Factor (IHF) sites, are preferentially recognized at the minimum membrane concentration at which oriC plasmid replication is inhibited in vitro. We have identified the putative proteins involved in hemimethylated oriC binding and cloned one of the corresponding genes (hobH). The purified LacZ-HobH fusion protein specifically binds oriC DNA at the same preferential sites as the membrane. A mutant of the hobH gene reveals partial asynchronous initiation of DNA replication.

Amino Acid Sequence↗
PubMed1999

CpG islands as genomic footprints of promoters that are associated with replication origins.

The primary target for DNA methylation in mammalian genomes is cytosine in the dinucleotide CpG. High densities of CpG dinucleotides are found in CpG islands, but paradoxically CpG islands are normally in a non-methylated state. Here, we speculate why CpG islands are immune to methylation and why they are so rich in guanine and cytosine relative to the surrounding DNA. We propose that CpG islands are associated with promoters that are transcriptionally active at totipotent stages of development and can also act as origins of DNA replication. CpG islands may be 'footprints' caused by early DNA replication intermediates at dual function promoters of this kind.

Animals↗
PubMed2000

Replication origin of Streptococcus pyogenes, organization and cloning in heterologous systems.

The origin of DNA replication (oriC) of Streptococcus pyogenes, group A streptococci (GAS), has been cloned in Escherichia coli and reintroduced by transformation into other GAS strains. Transformation frequencies into GAS strains with oriC-carrying plasmids occurred with unusually high frequencies. However, the oriC-containing plasmids in the new recipients were found to be unstable and had a tendency to integrate into the chromosome, even when a recA GAS strain was used as a recipient. The GAS oriC was able to direct the replication of autonomous plasmids in group B streptococcal recipients. The chromosomal organization of the oriC region of GAS relative to other bacterial species appears to be similar to oriC of Bacillus subtilis and other Gram-positive microorganisms.

Amino Acid Sequence↗
PubMed2002

A Functional chromatin domain does not resist X chromosome inactivation: silencing of cLys correlates with methylation of a dual promoter-replication origin.

To investigate the molecular mechanism(s) involved in the propagation and maintenance of X chromosome inactivation (XCI), the 21.4-kb chicken lysozyme (cLys) chromatin domain was inserted into the Hprt locus on the mouse X chromosome. The inserted fragment includes flanking matrix attachment regions (MARs), an origin of bidirectional replication (OBR), and all the cis-regulatory elements required for correct tissue-specific expression of cLys. It also contains a recently identified and widely expressed second gene, cGas41. The cLys domain is known to function as an autonomous unit resistant to chromosomal position effects, as evidenced by numerous transgenic mouse lines showing copy-number-dependent and development-specific expression of cLys in the myeloid lineage. We asked the questions whether this functional chromatin domain was resistant to XCI and whether the X inactivation signal could spread across an extended region of avian DNA. A generally useful method was devised to generate pure populations of macrophages with the transgene either on the active (Xa) or the inactive (Xi) chromosome. We found that (i) cLys and cGas41 are expressed normally from the Xa; (ii) the cLys chromatin domain, even when bracketed by MARs, is not resistant to XCI; (iii) transcription factors are excluded from lysozyme enhancers on the Xi; and (iv) inactivation correlates with methylation of a CpG island that is both an OBR and a promoter of the cGas41 gene.

Animals↗
PubMed1998

Multiple initiations in the c-myc replication origin independent of chromosomal location.

At supramolecular resolution, DNA synthesis begins at preferred replication origins in the chromosomes of metazoan cells. To characterize one of these origins in detail, the initiation of replication was examined in the HeLa c-myc origin. Polymerase chain reaction (PCR) amplification of size-fractionated nascent chromosomal DNAs revealed multiple replication initiation sites over a 12-kb region spanning the c-myc origin, including the transcribed region and the 5' and 3' flanking DNA of the gene. Two of the start sites for chromosomal replication occurred inside a 2.4-kb region of the origin that exhibits autonomously replicating sequence (ARS) activity. When a plasmid containing the 2.4-kb ARS region was transfected into HeLa cells, PCR mapping of nascent plasmid DNA confirmed that the plasmid replicated semiconservatively and autonomously and that replication did not initiate at random sites but rather began at multiple sites in a limited zone overlapping the c-myc DNA insert. Within the resolution of the PCR assay, the same sites that were used in the chromosomal c-myc origin were used in the 2.4-kb ARS fragment. The locations of replication start sites determined by PCR are considered in the context of other functional and structural elements of the c-myc origin.

Cell Polarity↗
PubMed1991

Critical sequences in the core of the P1 plasmid replication origin.

The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.

Bacterial Proteins↗
PubMed2004

Nonparametric methods for analyzing replication origins in genomewide data.

Due to the advent of high-throughput genomic technology, it has become possible to monitor cellular activities on a genomewide basis. With these new methods, scientists can begin to address important biological questions. One such question involves the identification of replication origins, which are regions in the chromosomes where DNA replication is initiated. One hypothesis is that their locations are nonrandom throughout the genome. In this article, we analyze data from a recent yeast study in which candidate replication origins were profiled using cDNA microarrays to test this hypothesis. We find no evidence for such clustering.

DNA, Complementary↗
PubMed1987

Transcripts within the replication origin, oriC, of Escherichia coli.

Transcription start and termination sites were mapped in the E. coli replication origin, oriC. Outward transcription from within oriC (promoters Pori-r and Pori-l) was found to start in vivo at position 178 for Pori-l and at positions 294 and 304 for Pori-r, respectively. These transcripts were terminated after 100-150 bases, at terminators designated Tori-l and Tori-r. Transcription from the 16 kd promoter, which lies clockwise adjacent to oriC and promotes transcription toward oriC, started at position 757 and gave transcripts with 3' ends at several positions within and to the left of the minimal replication origin. However, the majority of transcripts traversed the whole oriC region, and were not terminated within the DNA segment tested. Transcription of the chromosomal 16 kd gene was negatively regulated by DnaA protein and positively affected by dam methylation. The possible function of these transcripts is discussed.

DNA Replication↗
PubMed2005

Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.

Antineoplastic Agents, Alkylating↗
PubMed1992

Protein-DNA interactions at a yeast replication origin.

An understanding of the protein-DNA interactions in vivo at origins of DNA replication in eukaryotes is essential to delineate the mechanism of initiation of DNA synthesis and its control in the cell cycle. In the yeast Saccharomyces cerevisiae, a family of sequences known as autonomously replicating sequences (ARSs) function as origins of bidirectional DNA replication on plasmids and, in several instances, also in their normal chromosomal location. Here we use nucleotide resolution genomic footprinting to investigate the association of proteins with ARS1. Nuclease protection patterns indicate that at least two different cellular factors interact with functional elements in ARS1. The first seems to be ARS-binding factor 1. The second seems to be a novel protein that generates extensive protection over the essential ARS consensus sequence and phased DNaseI-sensitive sites across a functionally important flanking sequence. Hypersensitivity of this region to cleavage by copper phenanthroline indicates that it is under torsional strain, analogous to that produced at transcriptional start sites by assembly of an initiation complex. The protection in situ is similar to that generated by the origin recognition complex (ORC) protein.

Base Sequence↗
PubMed2005

Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection.

In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.

Cells, Cultured↗
PubMed1995

Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC.

Characterization of the proteins that interact with replication origins, as well as characterization of the mechanisms by which the levels and activities of these proteins are regulated during the cell cycle, is required to understand the initiation of chromosomal DNA replication in eukaryotic cells. We have previously shown that the first detectable step in the assembly of initiation complexes in vivo involves the binding of the multisubunit origin recognition complex (ORC) and the general transcription/replication factor ABF1 protein to origins. In this paper we show that ORC is present in cells at low levels, corresponding to little more than one complete complex per replication origin, indicating that in vivo origin recognition by ORC is extremely efficient. We show that this efficient recognition requires two sequence elements, the essential A element containing the ARS consensus sequence and the functionally important B1 element, both in vitro and in vivo. Moreover, we show that origin binding by ORC in vivo does not require any other functional sequence element, indicating that it occurs independently of the binding of other factors, such as ABF1. Our results suggest a model for the roles of the individual elements of yeast replication origins.

Base Sequence↗