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PubMed1995

A densely methylated DNA island is associated with a chromosomal replication origin in the human RPS14 locus.

We describe a 258-bp densely methylated DNA island (DMI) and chromosomal origin of bidirectional DNA replication within the transcribed portion of the human RPS14 intron 1. Together with the DMIs previously detected in two functional Chinese hamster replication origins [see Ref. 1, pp. 5636-5644], observations described in this report strengthen the correlation between densely methylated DNA islands and active mammalian chromosomal replication origins. Accordingly, DMIs may prove to be reliable physical markers for origins of bidirectional DNA replication in complex genomic DNAs of higher animals.

Animals↗
PubMed2005

Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome.

Cloning the whole 3.5-megabase (Mb) genome of the photosynthetic bacterium Synechocystis PCC6803 into the 4.2-Mb genome of the mesophilic bacterium Bacillus subtilis 168 resulted in a 7.7-Mb composite genome. We succeeded in such unprecedented large-size cloning by progressively assembling and editing contiguous DNA regions that cover the entire Synechocystis genome. The strain containing the two sets of genome grew only in the B. subtilis culture medium where all of the cloning procedures were carried out. The high structural stability of the cloned Synechocystis genome was closely associated with the symmetry of the bacterial genome structure of the DNA replication origin (oriC) and its termination (terC) and the exclusivity of Synechocystis ribosomal RNA operon genes (rrnA and rrnB). Given the significant diversity in genome structure observed upon horizontal DNA transfer in nature, our stable laboratory-generated composite genome raised fundamental questions concerning two complete genomes in one cell. Our megasize DNA cloning method, designated megacloning, may be generally applicable to other genomes or genome loci of free-living organisms.

Bacillus subtilis↗
PubMed1985

A model of bacterial DNA segregation based upon helical geometry.

A new mechanism to segregate daughter genomes in bacterial cells is suggested that is based upon the rules of geometry governing the helix clock (Mendelson, 1982a). The reorientation of cell surface string arrays used as a timing reference in the helix clock is capable of drawing apart the initial products of DNA replication. Physically linking the sister DNA replication origins to the ends of the initial cell surface string inserted into the cell surface at the start of a helix clock cycle, and linking the DNA terminus to a point along the length of the same string provides a means to mark the locations to which the genomes will segregate as well as the place where cell division will occur. The parallel packing of additional cell surface strings into an array which includes the string to which DNA is attached provides the necessary spatial rearrangements. The helical segregation model can account for the precise registration of cell divisions with the completion of replication forks in a multifork replication system, provides a basis for determining the relationship of sister cell sizes at division, and can also accommodate the asymmetrical divisions associated with minicell production and sporulation. Examination of the helical segregation theory under multifork DNA replication conditions moreover reveals that adjacent helical clocks are physically linked to one another although totally independent in terms of their progression through the clock cycle. A relationship between the initiation of DNA replication forks and the insertion of the first cell surface string associated with the start of a helix clock cycle is predicted by the model.

Cell Division↗
PubMed1987

Recombinant DNA in filamentous fungi: progress and prospects.

Recombinant DNA technology enables the creation of well-defined alterations in the genetic material of an organism. Methods to manipulate recombinant DNA in the filamentous fungi (a group of microorganisms that includes species of academic as well as commercial interest) have recently been developed. This has been the result of adaptation of procedures successfully employed in the manipulation of other microorganisms. There are a number of similarities in the behavior of recombinant DNA in different fungi, but a number of differences have also been observed between the filamentous and the nonfilamentous fungi. Such differences include the ability to identify DNA replication origins and the host range of expression of fungal genes.

Cloning, Molecular↗
PubMed2026

Origin flexibility governs robust ssDNA engagement by the DnaA initiator.

In model bacteria, initiation of chromosome replication requires engagement of single-stranded DNA by oligomers of the DnaA-family initiator assembled within the origin DNA. Although arrays of double-strand motifs recognized by DnaA are a general feature of the origins, the DnaA-binding single-strand elements are elucidated in only a limited number of species, and the mechanical principles governing their recognition remain elusive. Using the Alphaproteobacterium Caulobacter crescentus, we identify a previously uncharacterized GA-rich single-stranded element in the origin that directly engages DnaA oligomers and is essential for robust initiation. This element is positioned at a subkilobase distance from the DnaA oligomerization region and is brought into proximity through dynamic structural rearrangements. Moreover, DnaA oligomers exhibit an unexpectedly broad yet constrained capacity to accommodate single-stranded sequence variation. These findings provide the molecular basis for origin plasticity, highlighting how origins can diverge while preserving initiation logic.

DNA, Single-Stranded↗
PubMed1977

Origin of replication, oriC, of the Escherichia coli chromosome: mapping of genes relative to R.EcoRI cleavage sites in the oriC region.

A precise genetic-physical map of the tna-ilv region at 82 min on the genetic map of E. coli is obtained through deletion mapping and analysis by restriction endonuclease EcoRI of plasmids, derived from an F' carrying the genes between aroE and ilv. A locus, designated het, which in its diploid state results in slow growth and heterogeneity of cell size due to distorted cell division, maps between bglB and asn, 30-45 kb counterclockwise of ilv. The pattern of R.EcoRI cleavage sites in the het region is identical with the pattern obtained by Marsh and Worcel (1977) who analyzed DNA labeled preferentially in the region of the DNA replication origin (oriC). We suggest that oriC is identical with the het site and that it can be allocated to a position 32 kb counterclockwise of the ilv operon.

Chromosome Mapping↗
PubMed1984

Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA.

Polyomavirus large T antigen binds specifically to multiple sites within the regulatory region of the viral genome. Experiments done with crude extracts from wild-type virus-infected mouse cells and immunoprecipitation of protein-DNA complexes localized two high-affinity binding sites on the early region side of the DNA replication origin. Purification of the large T antigen by immunoaffinity chromatography made it possible to refine the analysis through application of DNase I footprinting. The high-affinity interactions were resolved into three closely spaced, but distinct, binding regions. These begin at a site only slightly overlapping the early boundary of the core replication origin, a location highly homologous to that of simian virus 40 large T antigen-binding site I, but then extend away from the origin toward the early coding sequence and thus span the early region transcriptional initiation sites. Each tight-binding region contains from two to four copies of the sequence 5'-(A = T)G(A greater than G)GGC-3' repeated at 9- to 11-base-pair spacing. At high protein concentrations and at low ionic strength, additional sites within the core replication origin and in the enhancer region were protected from DNase I digestion. These minor binding sites also included repeats of sequences related to the consensus, but at different spacings. Our results suggest that, unlike simian virus 40 DNA, the polyomavirus genome may have distinct regions of interaction with its large T antigen which separately are involved in initiation of DNA replication and the regulation of viral transcription.

Antigens, Viral, Tumor↗
PubMed1994

Identification of the primary structure and the coding capacity of the genome of insect iridescent virus type 6 between the genome coordinates 0.310 and 0.347 (7990 bp).

The primary structure and the coding capacity of the insect iridescent virus type 6--Chilo iridescent virus (CIV)--were determined between the genome coordinates 0.310 (EcoRI site) and 0.347 (ClaI site). The EcoRI CIV DNA fragment M (7.1 kb; 0.310-0.345 map units) harbors one out of at least six loci of DNA replication origins which is located at nucleotide position 485-513. The identification of the structural properties and the coding capacity of the EcoRI CIV DNA fragment M was carried out by DNA nucleotide sequencing, computer-aided sequence analysis and DNA/RNA hybridization. The EcoRI CIV DNA fragment M (7,099 bp; 71.14% A+T and 28.86% G+C) possesses two clusters of five tandemly organized repetitive DNA elements with complex structural arrangements (R1-R5) which are located between nucleotide positions 3272-3350 and 3403-3414. The analysis of the DNA sequences of the EcoRI CIV DNA fragment M revealed the presence of six open reading frames (ORFs 1-6). Two out of six detected putative proteins are of particular interest. ORF-2 was found to be terminated at nucleotide position 366 (TAA) within the DNA sequence of the EcoRI CIV DNA fragment L (0.345-0.381 map units; 7.4 kb). The analysis of ORF-2 (1,051 amino acids; 120 kD) revealed homologies to several DNA-directed RNA polymerases. ORF-6 encodes a protein (606 amino acids; 69 kD) which is related to a group of yeast, Drosophila and mammalian proteins of a distinct family of putative DNA and/or RNA helicases belonging to the 'DEAD/H' superfamily. The transcriptional activity of the EcoRI CIV DNA fragment M was determined by DNA/RNA hybridization experiments. These analyses revealed the existence of three RNA transcripts of about 3.4 kb (t1), 1.8 kb (t2) and 1.2 kb (t3) which agree with the predicted size of the expected RNA transcripts from ORF-2 (1,051 amino acids; 3.1 kb) and ORF-6 (606 amino acids; 1.8 kb).

Amino Acid Sequence↗
PubMed1995

Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.

New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit.

Animals↗
PubMed1999

Posttranscriptional regulation of US11 in cells infected with a herpes simplex virus 1 recombinant lacking both 222-bp domains containing S-component origins of DNA synthesis.

The US11 gene of herpes simplex virus 1 maps in the unique sequences of the short component of the HSV-1(F) genome approximately 775 bp from the center of the DNA replication origin (OriS) and encodes a virion protein which binds RNA in sequence- and conformation-specific fashion, negatively regulates the accumulation of a prematurely terminated transcript of UL34, associates in the infected cell with the 60S ribosomal subunit, and, late in infection, accumulates in nucleoli. We report the following: (i) Deletion of a 222-bp sequence including OriS (DeltaOriS) negatively affected the accumulation of the US11 protein without decreasing the accumulation of the US11 transcript. (ii) The defect, observed at all times after infection, was multiplicity independent, was unrelated to US11 protein stability, and apparently resulted from a cis-acting element since a coinfecting virus was unable to complement the DeltaOriS virus. (iii) Transcription from the US11 promoter initiated from three sites on the DeltaOriS virus. Transcripts initiated from two of the three initation sites accumulated similarly in cells infected with the DeltaOriS virus or wild-type parent virus. The low-abundance transcript initiating from the third site was apparently unique to the DeltaOriS virus but was not expected to alter the coding capacity of the mRNA. (iv) Infected cells accumulated RNA derived by antisense transcription of the genome domain containing the US11 gene. One transcript accumulated in larger amounts in cells infected with the DeltaOriS virus than in cells infected with parent or repaired virus.

Autoradiography↗
PubMed1993

Structural organization of the maxicircle variable region of Trypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites.

The maxicircle of the parasitic protozoan Trypanosoma brucei, one component of the mitochondrial genome, has size differences among isolates that localize to the variable region (VR) between the ND5 and 12S rRNA genes. We present here the nucleotide sequence of this entire region, thus completing the sequence of the maxicircle genome. We also find heterogeneously sized transcripts from throughout most of the VR. The VR has three distinct sections, each with characteristic repeated sequences. The repeated sequences in two sections are short and highly reiterated; the intraspecies size variation occurs within this region. The third section contains non-repetitive sequences and a large duplication immediately upstream of the 12S rRNA gene. Two repeat units within section I contain a sequence that has homology to the DNA replication origin of minicircles. This region also contains sequences with homology to topoisomerase II binding and cleavage sites. These findings suggest a role for the VR in DNA replication of the maxicircle.

Animals↗
PubMed2002

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to oris, an origin of viral DNA replication.

We have identified cellular proteins that interact with the herpes simplex virus type 1 (HSV-1) origin-binding protein (UL9 protein) by screening a HeLa cell complementary DNA library by using the yeast two-hybrid system. Approximately 7 x 10(5) colonies were screened. Five of the 48 positive clones contained cDNAs that encoded the p150(Glued) component of the dynactin complex, three contained cDNAs for the neural F Box 42-kDa protein (NFB42), which is highly enriched in neural tissue, and three contained hTid-1, a human homologue of the bacterial DnaJ protein. We have focused in this report on the interaction of the viral UL9 protein with the cellular hTid-1. In vitro immunoprecipitation experiments confirmed that hTid-1 interacts with the UL9 protein. Electrophoretic mobility-shift assays indicated that the hTid-1 enhances the binding of UL9 protein to an HSV-1 origin, ori(s), and facilitates formation of the multimer from the dimeric UL9 protein. hTid-1 had no effect on the DNA-dependent ATPase or helicase activities associated with the UL9 protein. These findings implicate hTid-1 in HSV-1 DNA replication, and suggest that this cellular protein may provide a chaperone function analogous to the DnaJ protein in Escherichia coli DNA replication.

Adenosine Triphosphatases↗
PubMed1981

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments.

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).

Cloning, Molecular↗
PubMed2006

Mechanism of DNA recognition at a viral replication origin.

Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1452-641, to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol-1, with 11.9 kcal mol-1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1452-641.DNA complex is accompanied by a heat capacity change of -1.22 kcal mol-1 K-1, a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1.DNA complex conformers are in slow equilibrium; one dissociates slowly (t1/2 approximately 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1/2 approximately 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6x10(8) m-1 s-1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of approximately 2.0 s-1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.

Animals↗
PubMed1983

The mitochondrial genome of Saccharomyces cerevisiae contains numerous, densely spaced autonomously replicating sequences.

Restriction fragments produced by a complete Sau3A cleavage of Saccharomyces cerevisiae grande mitochondrial DNA were ligated into the yeast-Escherichia coli shuttle vector YIp5 to establish a clone library representing the mitochondrial genome. 30 hybrid plasmids with an average insert size of 1200 bp were chosen at random and tested for the presence of an autonomously replicating sequence (ars). Over two-thirds of these plasmids transformed yeast at high frequency, indicating the mitochondrial genome contains a large number of ars elements. Our calculations suggest there may be over 40 ars elements contained within the mitochondrial DNA with an average spacing of less than 1700 bp. Mapping experiments indicate that ars elements can be found at many locations on the mitochondrial genome, and in the initial example we have tested, the locations of ars elements derived from grande and petite mtDNAs appear to coincide. If we assume that these ars elements represent mitochondrial DNA replication origins used in vivo, these observations would explain in part the fact that petite mtDNAs can be derived from any location on the grande mitochondrial genome.

Chromosome Mapping↗
PubMed2003

Sequence-independent DNA binding and replication initiation by the human origin recognition complex.

We report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication.

Animals↗
PubMed2004

The regulation of competence to replicate in meiosis by Cdc6 is conserved during evolution.

DNA replication licensing is an important step in the cell cycle at which cells become competent for DNA replication. When the cell cycle is arrested for long periods of time, this competence is lost. This is the case for somatic cells arrested in G0 or vertebrate oocytes arrested in G2. CDC6 is a factor involved in replication initiation competence which is necessary for the recruitment of the MCM helicase complex to DNA replication origins. In Xenopus, we have previously shown that CDC6 is the only missing replication factor in the oocyte whose translation during meiotic maturation is necessary and sufficient to confer DNA replication competence to the egg before fertilization (Lemaitre et al., 2002: Mol Biol Cell 13:435-444; Whitmire et al., 2002: Nature 419:722-725). Here, we report that this oogenesis control has been acquired by metazoans during evolution and conserved up to mammals. We also show that, contrary to eukaryotic metazoans, in S. pombe cdc18 (the S. pombe CDC6 homologue), CDC6 protein synthesis is down regulated during meiosis. As such, the lack of cdc18 prevents DNA replication from occurring in spores, whereas the presence of cdc6 makes eggs competent for DNA replication.

Animals↗
PubMed1991

The repression of the reverse-oriented transcription from the adenovirus terminus by NFI in competition with TFIID.

Nuclear factor I (NF) represses the transcription which is promoted by the cloned adenovirus (Ad) type 5 DNA replication origin and is reverse-oriented with respect to the direction of the replication. The mechanism of this repression by NFI was investigated. In the cell-free transcription system, the repression was observed only when NFI was present during the formation of the transcription initiation complex. From the results of DNase I protection experiments, it was indicated that NFI bound to its binding site in the Ad replication origin prevents TFIID from proper binding to the adjacent AT-rich region and consequently represses the transcription.

Adenoviridae↗