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At least 1,423 records · Page 79Linked to original sources
PubMed1986

Transcription termination within the Escherichia coli origin of DNA replication, oriC.

Initiation of DNA replication from the Escherichia coli origin, oriC, is dependent on an RNA polymerase-mediated transcription event. The function of this RNA synthetic event in initiation, however, remains obscure. Since control of the synthesis of this RNA could serve a key role in the overall initiation process, transcription regulatory sites within and near oriC were identified using the galK fusion vector system. Our results confirm the existence of a transcription termination signal within oriC, first identified by Hansen et al. (1981), for the 16 kd transcript that is transcribed counterclockwise towards oriC. Termination is shown to be 92% efficient. A similar approach led to the detection of transcription termination within the chromosomal replication origin of Klebsiella pneumoniae. Approximately 50% of the E. coli 16 kd transcripts appear to terminate before reaching oriC between the XhoI (+416 bp) and the HindIII (+243 bp) sites. The predominant 3' ends of RNA that enter oriC, as determined by SI nuclease mapping, were located at positions +20 +/- 2, +23 +/- 2, +37, +39, +52, +66, +92, and +107. These termination sites, which map cl to RNA . DNA junctions identified by Kohara et al. (1985), appear as triplets and quadruplets. The E. coli oriC Pori-L promoter described in in vitro transcription studies by Lother and Messer (1981) was not detected in this study in either wildtype cells or isogenic dnaA mutants at the nonpermissive temperature. A new promoter activity, Pori-R1, was identified within the E. coli origin in the clockwise direction.

DNA Replication↗
PubMed2000

A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation.

SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery.

Bacterial Outer Membrane Proteins↗
PubMed1984

Multiple initiation sites of DNA replication flanking the origin region of lambda dv genome.

Early replicative intermediates of lambda dv plasmid were prepared by an in vitro replication system in the presence of 2',3'-dideoxycytidine 5'-triphosphate, an inhibitor of DNA chain elongation. Short-chain DNAs produced from regions near the replication origin were purified from the intermediates. A fraction of the DNAs was covalently linked to primer RNA. The transition sites from primer RNA to DNA synthesis were mapped along the nucleotide sequence of the genome, by eliminating the RNA by alkaline hydrolysis and labeling the freshly exposed 5' ends of DNA with 32P. The transition sites were found to be located on both sides of the ori region, which includes four 19-base-pair repeats where one of the lambda specific initiator proteins, O, binds. No transition arose within the ori region. The transition sites are multiple on both sides of the ori region and are clustered in one of the two strands in such a way that DNA syntheses from the two sides converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis, reflecting the asymmetric bidirectional replication of lambda dv DNA.

Bacteriophage lambda↗
PubMed1986

The gene II proteins of the filamentous phages IKe and Ff (M13, fd and f1) are not functionally interchangeable during viral strand replication.

Gene II protein is the only phage-encoded protein required for the double-stranded DNA replication of the distantly related filamentous phages IKe and Ff (M13, fd and f1). Complementation studies have demonstrated that, despite a significant degree of homology between the nucleotide sequences of the gene II's of IKe and Ff and the core's (domains A) of their viral strand replication origins, the biological functions of the gene II proteins are not interchangeable. The specificity of these proteins is not determined by the nucleotide sequence (domain B) which is required for efficient initiation of viral strand replication of Ff. In fact, our data indicate that a sequence with a similar function as domain B in Ff does not form part of the viral strand replication origin of IKe.

Base Sequence↗
PubMed1981

Role of RNA transcripts in replication incompatibility and copy number control in antibiotic resistance plasmid derivatives.

The genes required for autonomous replication and incompatibility in the antibiotic resistance plasmids R100 and R1 have been located within a 2.5-kilobase region of the 90-kilobase genome, within which the incompatibility gene occupies a 1.3-kilobase region excluding the replication origin. We now report that three RNA species are synthesized in vitro from the 2.5-kilobase region, which R100 and R1 have in common. One, a long RNA molecule which is transcribed in the direction of DNA replication, probably acts as a messenger or a protein required for plasmid replication. The second RNA species, only 91 nucleotides long, is transcribed in the opposite direction, from a region of the DNA entirely contained within the first and known to specify incompatibility and copy control functions. The third RNA species, 150 bases long, is transcribed from a region including the replication origin; it may be a primer of DNA synthesis or, in conjunction with the second of the three RNA species, an influence in the control of replication.

Chromosome Mapping↗
PubMed2004

The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation.

Before S phase, cells license replication origins for initiation by loading them with Mcm2-7 heterohexamers. This process is dependent on Cdc6, which is recruited to unlicensed origins. Using Xenopus egg extracts we show that although each origin can load many Mcm2-7 hexamers, the affinity of Cdc6 for each origins drops once it has been licensed by loading the first hexamers. This encourages the distribution of at least one Mcm2-7 hexamer to each origin, and thereby helps to ensure that all origins are licensed. Although Cdc6 is not essential for DNA replication once licensing is complete, Cdc6 regains a high affinity for origins once replication forks are initiated and Mcm2-7 has been displaced from the origin DNA. We show that the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition. These results show that Cdc6 plays multiple roles in ensuring precise chromosome duplication.

Animals↗
PubMed1987

A nuclease-hypersensitive region forms de novo after chromosome replication.

Regular nucleosome arrays in eucaryotic chromosomes are punctuated at specific locations, such as active promoters and replication origins, by apparently nucleosome-free sites, also called nuclease-hypersensitive, or exposed, regions. The -400-base pair-exposed region within simian virus 40 (SV40) chromosomes is present in approximately 20% of the chromosomes in lytically infected cells and encompasses the replication origin, transcriptional enhancer, and both late and early SV40 promoters. We report that nearly all SV40 chromosomes lacked the exposed region during replication and that newly formed chromosomes acquired the exposed region of the same degree as did bulk SV40 chromosomes within 1 h after replication. Furthermore, a much lower but significant level of exposure was detectable in late SV40 replication intermediates, indicating that formation of the exposed region could start within minutes after passage of the replication fork.

Chromatin↗
PubMed1994

The initiation cascade for chromosome replication in wild-type and Dam methyltransferase deficient Escherichia coli cells.

'Newborn' Escherichia coli B/r cells, obtained by membrane elution, were used to study the cell cycles of wild-type and Dam methyltransferase mutants. In wild-type cells, initiation of chromosome replication was synchronous and tightly controlled. In dam mutants, initiation was altered, but not random. We propose that this is due to the absence of an initiation cascade caused by liberated DnaA molecules, and that this cascade normally synchronizes initiation. The dam- cells contained mainly two, three or four replication origins, and this affected nucleoid partitioning as well as cell division. In cultures growing with a 50 min doubling time, a variety of cell cycles were present and half the origins were used every 25 min. Some cells had a 25 min interdivision time, whereas others had an interdivision time longer than the generation time. Partitioning of nucleoids containing unequal numbers of replication origins could also be readily observed by fluorescence microscopy in the dam mutant. Based upon these observations we propose that the dam mutant is also an initiation cascade mutant.

Bacterial Proteins↗
PubMed2006

Nonparametric inference for local extrema with application to oligonucleotide microarray data in yeast genome.

Identifying local extrema of expression profiles is one primary objective in some cDNA microarray experiments. To study the replication dynamics of the yeast genome, for example, local peaks of hybridization intensity profiles correspond to putative replication origins. We propose a nonparametric kernel smoothing (NKS) technique to detect local hybridization intensity extrema across chromosomes. The novelty of our approach is that we base our inference procedures on equilibrium points, namely those locations at which the first derivative of the intensity curve is zero. The proposed smoothing technique provides both point and interval estimation for the location of local extrema. Also, this technique can be used to test for the hypothesis of either one or multiple suspected locations being the true equilibrium points. We illustrate the proposed method on a microarray data set from an experiment designed to study the replication origins in the yeast genome, in that the locations of autonomous replication sequence (ARS) elements are identified through the equilibrium points of the smoothed intensity profile curve. Our method found a few ARS elements that were not detected by the current smoothing methods such as the Fourier convolution smoothing.

Biometry↗
PubMed1995

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.

Adenosine Triphosphate↗
PubMed1996

In vivo topoisomerase II cleavage sites in the ribosomal DNA of Physarum polycephalum.

We have analyzed the topoisomerase II cleavage sites in the extrachromosomal ribosomal DNA of the lower eukaryote Physarum polycephalum using the topoisomerase II-specific inhibitor, 6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid. Most of the in vivo topoisomerase II cleavage sites were found either in the transcribed region of ribosomal DNA or in the palindromic region surrounded by the replication origins. Two classes of sites were identified: those which correlate with DNase I hypersensitive sites and corresponding to an open chromatin configuration (transcribed region) and internucleosomal cleavage sites (in the region of replication origins). Topoisomerase II drug-induced cleavage in the ribosomal DNA was considerably reduced upon Physarum differentiation to a dormant stage of life, the spherules. In contrast, the amount of drug-dependent cleavage was found to increase during the metaphase of mitosis, when rDNA transcription is shut off. These findings suggest a role for topoisomerase II in the ribosomal DNA minichromosomes segregation, in addition to its role in transcription. Finally, the similarity between in vivo sites and those observed following drug treatment of isolated nuclei indicates that no profound change occurs in rDNA chromatin conformation during nuclei isolation. By contrast, in vitro cleavage sites with purified topoisomerase II weakly correlate to in vivo, indicating a prominent role for chromatin structure in determining the interaction sites of topoisomerase II with DNA in vivo.

Animals↗
PubMed1990

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.

Animals↗
PubMed1995

A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence.

Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5' end of the rG region. The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required. The efficiency of hybrid formation depends on the length of RNA synthesized 5' to CSBII and the type of RNA polymerase employed. Once made, the RNA strand of an RNA-DNA hybrid can serve as an effective primer for mitochondrial DNA polymerase. These results reveal a new mechanism for persistent RNA-DNA hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA hybrid.

Base Sequence↗
PubMed1995

Mycobacterium smegmatis dnaA region and autonomous replication activity.

Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element. As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene. Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5' end of the dnaN gene, which codes for the beta subunit of DNA polymerase III. Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M. smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M. smegmatis. We suggest that the M. smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.

Amino Acid Sequence↗
PubMed2004

Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination.

In late mitosis and G1, a complex of the essential initiation proteins Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other times licensing is inhibited by cyclin-dependent kinases (CDKs) and geminin, thus ensuring that origins fire only once per cell cycle. Here we show that, paradoxically, CDKs are also required to inactivate geminin and activate the licensing system. On exit from metaphase in Xenopus laevis egg extracts, CDK-dependent activation of the anaphase-promoting complex (APC/C) results in the transient polyubiquitination of geminin. This ubiquitination triggers geminin inactivation without requiring ubiquitin-dependent proteolysis, and is essential for replication origins to become licensed. This reveals an unexpected role for CDKs and ubiquitination in activating chromosomal DNA replication.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1991

Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.

Animals↗
PubMed1995

Genetic analysis of the minimal replicon of plasmid pIP417 and comparison with the other encoding 5-nitroimidazole resistance plasmids from Bacteroides spp.

The nucleotide sequence of the DNA replication origin region of a Bacteroides vulgatus plasmid, pIP417, encoding 5-nitroimidazole resistance has been determined. This region of 1934 bp presents some characteristics similar to those of other replication protein-dependent origins. It contains a large open reading frame which could encode a basic Rep protein (RepA) of 36.8 kDa. Upstream of this ORF exist an AT-rich region, three direct repeats (iterons) of 21 bp, multiple DnaA binding sites, and sites, and sites for the integration host factor (IHF). Moreover, the amino acid sequence of the pIP417 RepA protein shows similarities with those of other Rep proteins encoded by plasmids of gram-negative bacteria: pRO1600 from Pseudomonas aeruginosa; pPS10 from Pseudomonas syringae; pFA3 from Neisseria gonorrhoeae; and two cryptic plasmids from Campylobacter hyointestinalis and Butyrivibrio fibrisolvens. Although RepA can be expressed in an Escherichia coli in vitro transcription-translation assay, vectors containing the pIP417 replication origin did not replicate in E. coli. The homology of the pIP417 replication region with the corresponding regions of other Bacteroides spp, plasmids was also studied by Southern blot hybridization. The results indicated that the repA gene of plasmid pIP417 is homologous to that of plasmid pIP421, but not of plasmid pIP419. The replication region of plasmid pIP421 was sequenced and showed about 80% identity at the nucleotide level with that of pIP417. A small (3634-bp) cloning vector (pFK12) of entirely defined nucleotide sequence was constructed for Bacteroides spp.

Amino Acid Sequence↗