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Results for “Origin Recognition Complex”

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At least 469 records · Page 26Linked to original sources
PubMed1998

The use of oriC-dependent phage infection to characterize the ultra violet (UV)-induced inhibition of initiation of DNA replication in Escherichia coli.

The oriC transducing phage lambda poriCc is a pseudovirulent phage capable of forming plaques on a lambda lysogen. This phenotype is dependent upon the presence of the oriC insert. The ability of lambda poriCc to form plaques on a lambda lysogen represents a potential phage assay system for studying aspects of oriC function. In the present study we establish that lambda poriCc infection of a lambda lysogen is a legitimate assay for oriC function. We use this assay to confirm the previously reported observation that initiation of DNA replication from oriC is transiently inhibited in a ultra violet (UV) irradiated cell at doses greater than 60 J/m2. We further demonstrate using this assay that the UV induced inhibition of initiation of DNA replication from oriC is not a SOS function nor a heat shock function. In the course of these studies, we found that lambda poriCc infection of a non-lysogenic cell is extremely sensitive to pre-irradiation of the Escherichia coli host. We postulate that the sensitivity of lambda poriCc replication to host cell pre-irradiation reflects in some way the transient inhibition of initiation of DNA replication from oriC following UV irradiation.

Bacteriophage lambda↗
PubMedDate not supplied

Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants.

BACKGROUND: The accurate duplication and packaging of the genome is an absolute prerequisite to the segregation of chromosomes in mitosis. To understand the process of cell-cycle chromosome dynamics further, we have performed the first detailed characterization of a mutation affecting mitotic chromosome condensation in a metazoan. Our combined genetic and cytological approaches in Drosophila complement and extend existing work employing yeast genetics and Xenopus in vitro extract systems to characterize higher-order chromosome structure and function. RESULTS: Two alleles of the ORC2 gene were found to cause death late in larval development, with defects in cell-cycle progression (delays in S-phase entry and metaphase exit) and chromosome condensation in mitosis. During S-phase progression in wild-type cells, euchromatin replicates early and heterochromatin replicates late. Both alleles disrupted the normal pattern of chromosomal replication, with some euchromatic regions replicating even later than heterochromatin. Mitotic chromosomes were irregularly condensed, with the abnormally late replicating regions of euchromatin exhibiting the greatest problems in mitotic condensation. CONCLUSIONS: The results not only reveal novel functions for ORC2 in chromosome architecture in metazoans, they also suggest that the correct timing of DNA replication may be essential for the assembly of chromatin that is fully competent to undergo mitotic condensation.

Animals↗
PubMed2003

Enigmatic variations: divergent modes of regulating eukaryotic DNA replication.

Proteins involved in DNA replication are conserved from yeast to mammals, suggesting that the mechanism was established at an early stage of eukaryotic evolution. In spite of this common origin, recent findings have revealed surprising variations in how replication initiation is controlled, implying that a conserved mechanism has not necessarily resulted in regulatory conservation.

Animals↗
PubMed2001

A ParE-ParC fusion protein is a functional topoisomerase.

Type II topoisomerases are responsible for DNA unlinking during DNA replication and chromosome segregation. Although eukaryotic enzymes are homodimers and prokaryotic enzymes are heterotetramers, both prokaryotic and eukaryotic type II topoisomerases belong to a single protein family. The amino- and carboxyl-terminal domains of eukaryotic enzymes are homologous to the ATP-binding and catalytic subunits of prokaryotic enzymes, respectively. Topoisomerase IV, a prokaryotic type II topoisomerase, consists of the ATP-binding subunit, ParE, and the catalytic subunit, ParC. We have joined the coding regions of parE and parC in frame and constructed a fusion protein of the two subunits of topoisomerase IV. This fusion protein, ParEC, can catalyze both decatenation and relaxation reactions. The ParEC protein is also capable of decatenating replicating daughter DNA molecules during oriC DNA replication in vitro. Furthermore, the fusion gene, parEC, complements the temperature-sensitive growth of both parC and parE strains, indicating that the ParEC protein can substitute for topoisomerase IV in vivo. These results demonstrate that a fusion protein of the two subunits of topoisomerase IV is a functional topoisomerase. Thus, a heterotetrameric type II topoisomerase can be converted into a homodimeric type II topoisomerase by gene fusion.

Artificial Gene Fusion↗
PubMed2002

Motors and switches: AAA+ machines within the replisome.

Clamp loaders are required to load the ring-shaped clamps that tether replicative DNA polymerases onto DNA. Recently solved crystal structures, along with a series of biochemical studies, have provided a detailed understanding of the clamp loading reaction. In particular, studies of the Escherichia coli clamp loader--an AAA+ machine--have provided insights into the architecture of clamp loaders from eukaryotes, bacteriophage T4 and archaea. Other AAA+ proteins are also involved in the initiation of DNA replication, and studies of the E. coli clamp loader indicate mechanisms by which these proteins might function.

Archaea↗
PubMed2005

Spotted-dick, a zinc-finger protein of Drosophila required for expression of Orc4 and S phase.

The highly condensed chromosomes and chromosome breaks in mitotic cells of a Drosophila mutant, spotted-dick/pita, are the consequence of defects in DNA replication. Reduction of levels of Spotted-dick protein, by either RNAi or mutation, leads to the accumulation of cells that have DNA content intermediate to 2N and 4N in proliferating tissues and also compromises endoreduplication in larval salivary glands. The Spotted-dick Zinc-finger protein is present in the nuclei of cells committed to proliferation but necessary in cells undertaking S phase. We show that Spotted-dick/Pita functions as a transcription factor and that, in cultured S2 cells, it is an activator of expression of some 30 genes that include the Orc4 gene, required for initiation of DNA replication. Chromatin immunoprecipitation indicates that it associates with the genes that it activates in S2 cells together with other sites that could represent genes activated in other tissues. We discuss the role of Spotted-dick in the coordination of cellular growth and DNA replication.

Animals↗
PubMed2002

Modulation of Mycobacterium tuberculosis DnaA protein-adenine-nucleotide interactions by acidic phospholipids.

The biochemical aspects of the initiation of DNA replication in Mycobacterium tuberculosis are unknown. To understand this process, we overproduced, purified and characterized the recombinant M. tuberculosis DnaA protein. The M. tuberculosis DnaA protein binds the origin of replication (oriC), ATP and ADP, and exhibited weak ATPase activity. ADP, after hydrolysis of ATP, remained strongly associated with DnaA and the exchange of ATP for bound ADP was weak. Vesicles prepared from acidic phospholipids, such as phosphatidylinositol, cardiolipin and phosphatidylglycerol, promoted dissociation of both ADP and ATP, whereas the neutral phospholipid phosphatidylethanolamine did not. The phospholipid-mediated dissociation of ATP was decreased in the presence of the M. tuberculosis oriC, whereas dissociation of ADP was stimulated in the presence of oriC. Acidic phospholipids in micelles, however, were not efficient in dissociating bound nucleotides from DnaA. Together, these results suggest that both polar head groups and membrane bilayer structure play an important role in M. tuberculosis DnaA-adenine-nucleotide interactions. We suggest that initiation of M. tuberculosis oriC involves intimate interactions between DnaA, adenine nucleotides and membrane phospholipids, and the latter helps to ensure that only the ATP form of the DnaA protein interacts continuously with oriC.

Adenine Nucleotides↗
PubMed2000

Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli.

BACKGROUND: We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions. RESULTS: The present study shows that (i) appearance of SeqA foci depends on continuous DNA replication, suggesting that the SeqA foci represent clusters consisting of SeqA and newly replicated hemimethylated DNA, (ii) in a synchronous round of replication, a single SeqA focus at midcell separates into two foci and these foci abruptly migrate in opposite directions midway through replication from oriC to the terminus, and (iii) oriC is replicated at midcell but replicated oriC copies remain linked with each other at midcell for 40 min after replication at 30 degrees C. Subsequently, the linked oriC copies separate and migrate gradually towards both borders of the nucleoid before cell division. CONCLUSIONS: A single cluster of SeqA-bound hemimethylated DNA segment separates into two clusters and these clusters migrate abruptly in a bipolar fashion during progress of replication and prior to separation of linked sister oriC copies.

Bacterial Outer Membrane Proteins↗
PubMed1998

Are minichromosomes valid model systems for DNA replication control? Lessons learned from Escherichia coli.

Initiation of chromosome replication is a key event in the life cycle of any organism. Little is known, however, about the regulatory mechanisms of this vital process. Conventionally, the initiation mechanism of chromosome replication in microorganisms has been studied using plasmids in which an origin of chromosome replication has been cloned, rather than using the chromosome itself. The reason for this is that even bacterial chromosomes are so large that biochemical and genetic manipulations become difficult and cumbersome. Recently, the combination of flow cytometry and genetic methods, in which modifications of the replication origin are systematically introduced onto the chromosome, has made possible detailed studies of the molecular events involved in the control of replication initiation in Escherichia coli. The results indicate that requirements for initiation at the chromosomal origin, oriC, are drastically different from those for initiation at cloned oriC.

Chromosomes, Bacterial↗
PubMed2001

CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli.

CspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of beta-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA. We propose that CspD dimers, with two independent beta-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.

Amino Acid Sequence↗
PubMed2002

Eclipse period without sequestration in Escherichia coli.

The classical Meselson-Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy ((15)NH(4)+ and (13)C(6)-glucose) medium were shifted to light ((14)NH(4)+ and (12)C(6)-glucose) medium. The HH-, HL- and LL-DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene-specific probes. The eclipse period, estimated from the kinetics of conversion of HH-DNA to HL- and LL-DNA, turned out to be 0.60 generation times for the wild-type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Bacterial Outer Membrane Proteins↗
PubMed2002

Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis.

Current views of bacterial chromosome segregation vary in respect of the likely presence or absence of an active segregation mechanism involving a mitotic-like apparatus. Furthermore, little is known about cis-acting elements for chromosome segregation in bacteria. In this report, we show that two separate DNA regions, a 3' coding region of dnaA and the AT-rich sequence between dnaA and dnaN (the initial opening site of duplex DNA during replication), are necessary for efficient segregation of the chromosome in Bacillus subtilis. When a plasmid replicon was integrated into argG, far from oriC, on the chromosome and then the oriC function was disrupted, the oriC-deleted mutant formed anucleate cells at 5% possibly because of defects in chromosome segregation. However, when the two DNA sequences were added near oriN, frequency of anucleate cells decreased to 1%. In these cells, the origin (argG) regions were localized near cell poles, whereas they were randomly distributed in cells without the two DNA sequences. These results suggest that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B. subtilis.

Bacillus subtilis↗