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At least 523 records · Page 29Linked to original sources
PubMed1984

Sequence of the putative origin of replication in the UL region of herpes simplex virus type 1 ANG DNA.

Interest has been stimulated concerning the region mapping between 0.38 and 0.42 on the prototype configuration of the herpes simplex virus type 1 (HSV-1) genome due to the high probability of the presence there of a second origin of DNA replication. A 960 bp restriction fragment (HinfI E) of a class II defective HSV-1 ANG DNA has been sequenced using the viral DNA rather than molecularly cloned DNA. This fragment includes the BamHI U/R cleavage site, mapping at approximately 0.4. Part of the sequence derived in this study displays homology with the origins of DNA replication contained in TRS/IRS of HSV-1 and HSV-2 DNA. The homologous region comprising 76 bp occurs as two copies, each of which contains two palindromically arranged copies of an 8 bp sequence identical to the 'consensus' sequence reported to be part of the origin of DNA replication at the terminus of the mammalian adenoviruses. It can be deduced from a comparison of this structure to the TRS/IRS origin of HSV-1 and HSV-2 that there are two origins of replication in the UL region of HSV-1 ANG DNA. Assuming that the orientation of the consensus sequence is relevant to the direction of DNA replication, one can conclude that the UL origin(s) of HSV-1 ANG is (are) bidirectional. It has not yet been possible to clone DNA fragments molecularly which include the region spanning the UL origin(s) of HSV-1 DNA.

Base Sequence↗
PubMed1982

Initiation of replication at specific origins in DNA molecules microinjected into unfertilized eggs of the frog Xenopus laevis.

Initiation of DNA replication at specific origins was observed by electron microscopy after microinjection of pXlr11, pXlr14 or Col E1 plasmid DNA molecules into unfertilized eggs of the frog, Xenopus laevis. These results are in apparent contradiction with published reports (Harland and Laskey, Cell 21, 761-771, 1980; Laskey and Harland, Cell 24, 283-284, 1981) that specific origin sites were not used in Xenopus laevis eggs. We suggest that eucaryotic origins exist that both increase the probability of replication of contiguous sequences and determine the site at which replication is most likely to begin.

Animals↗
PubMed2004

Sequence requirements for function of the Drosophila chorion gene locus ACE3 replicator and ori-beta origin elements.

The developmentally regulated amplification of the Drosophila third chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-beta is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the approximately 1.2 kb S18 chorion gene and the 840 bp ori-beta. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-beta, thereby confirming the sequence specificity of ori-beta. Deletion mapping of ori-beta identified two required components: a 140 bp 5' element and a 226 bp A/T-rich 3' element called the beta-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2) recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satin eliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminated foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-beta nucleate the formation of a ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon chiffon.

Animals↗
PubMed1989

Tracking bacterial DNA replication forks in vivo by pulsed field gel electrophoresis.

The location of chromosomal DNA replication forks was identified in synchronously replicating E. coli cultures by pulse labeling DNA at specific times with 14C-thymidine and following incorporation of radionucleotide into genomic Not I restriction fragments. This technique could be used to characterize chromosomal DNA replication, to characterize mutations which affect this process, to identify the location of DNA replication origins and termini as well as aid in the construction of macrorestriction maps. Here, we further characterize the DNA replication mutations divE and dnaK and preliminary characterize the genomic organization of E. coli isolate 15.

Chromosome Mapping↗
PubMed1994

Association of origin binding protein and single strand DNA-binding protein, ICP8, during herpes simplex virus type 1 DNA replication in vivo.

The herpes simplex virus type 1 (HSV-1) origin binding protein (UL9 protein) interacts specifically with the HSV-1 encoded single strand DNA-binding protein ICP8 (Boehmer, P.E. and Lehman, I.R. (1994) Proc. Natl. Acad. Sci. U.S.A. 90, 8444-8448). A UL9 mutant protein (UL9DM27) that lacks the C-terminal 27 amino acids shows normal origin-specific DNA binding and retains its DNA-dependent ATPase and helicase activities, but has a greatly reduced affinity for ICP8. The extreme C-terminal portion of the UL9 protein is therefore required for ICP8 binding. The helicase activity of the UL9DM27 protein is approximately 8-fold greater than that of the wild type UL9 protein and is not stimulated by ICP8. The UL9DM27 protein has a reduced ability to replicate origin-containing plasmids in vivo. Consequently, the interaction between the UL9 protein and ICP8 is likely to be important for origin-dependent DNA replication in vivo, presumably to promote efficient unwinding of the DNA at an HSV-1 origin of DNA replication.

Amino Acid Sequence↗
PubMed1999

Tamplicon-7, a novel T-lymphotropic vector derived from human herpesvirus 7.

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.

Base Sequence↗
PubMed1992

Structural and functional studies on phi 29 DNA polymerase.

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.

Amino Acid Sequence↗
PubMed2002

Identification of Tah11/Sid2 as the ortholog of the replication licensing factor Cdt1 in Saccharomyces cerevisiae.

Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Amino Acid Sequence↗
PubMed1993

Interactions of USF and Ku antigen with a human DNA region containing a replication origin.

By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin. The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins. The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF. The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen. In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends. The potential role of B48 binding site and of these cognate proteins in origin activation is discussed.

Amino Acid Sequence↗
PubMed2001

Adaptive reversions of a frameshift mutation in arrested Saccharomyces cerevisiae cells by simple deletions in mononucleotide repeats.

Adaptive mutations are characterised as the outcome of an as yet unknown mechanism, which allows a few individuals of a cell population to overcome a starvation-induced cell cycle arrest and to proliferate. A release from such a non-lethal growth limitation is accomplished by mutations generated without DNA replication. Originally adaptive mutations were described in Escherichia coli, but more recently also in a simple eukaryote, the budding yeast Saccharomyces cerevisiae. We are studying the adaptive reversion of a frameshift allele which occurs when an auxotrophic yeast strain is starved for the amino acid essential for its proliferation. In this communication, we report on the DNA sequences from the locus concerned. Comparison between sequences from revertant clones which arose several days after growth arrest by starvation and those from revertants produced during proliferation shows significantly different mutation spectra: for replication-dependent revertants nucleotide gains and losses in a variety of sequence contexts are reasonably balanced, whereas for the replication-independent, i.e. adaptive, revertants mainly simple deletions in mononucleotide repeats were observed. These mutations resemble those known to originate from DNA polymerase slippage errors which were miscorrected or had escaped correction by the mismatch repair machinery. Our data present strong evidence for differences in the mechanistic origins of adaptive versus DNA replication-dependent mutations in a eukaryote. Most probably, mutations in non-replicating cells contribute to evolution, and if conserved in mammals, to human carcinogenesis.

Base Sequence↗
PubMed1989

Replication origins of single-stranded-DNA plasmid pUB110.

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.

Base Sequence↗
PubMed2002

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein.

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.

Amino Acid Substitution↗
PubMed1998

Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant.

Mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae contains highly conserved sequences, called rep/ori, that are associated with several aspects of its metabolism. These rep/ori sequences confer the transmission advantage exhibited by a class of deletion mutants called hypersuppressive petite mutants. In addition, because they share features with the mitochondrial leading-strand DNA replication origin of mammals, rep/ori sequences have also been proposed to participate in mtDNA replication initiation. Like the mammalian origins, where transcription is used as a priming mechanism for DNA synthesis, yeast rep/ori sequences contain an active promoter. Although transcription is required for maintenance of wild-type mtDNA in yeast, the role of the rep/ori promoter as a cis-acting element involved in the replication of wild-type mtDNA is unclear, since mitochondrial deletion mutants need neither transcription nor a rep/ori sequence to maintain their genome. Similarly, transcription from the rep/ori promoter does not seem to be necessary for biased inheritance of mtDNA. As a step to elucidate the function of the rep/ori promoter, we have attempted to detect transcription-dependent DNA transactions in the mtDNA of a hypersuppressive petite mutant. We have examined the mtDNA of the well-characterized petite mutant a-1/1R/Z1, whose repeat unit shelters the rep/ori sequence ori1, in strains carrying either wild-type or null alleles of the nuclear genes encoding the mitochondrial transcription apparatus. Complex DNA transactions were detected that take place around GC-cluster C, an evolutionarily conserved GC-rich sequence block immediately downstream from the rep/ori promoter. These transactions are strictly dependent upon mitochondrial transcription.

DNA Replication↗
PubMed2001

Cell cycle: Flies teach an old dogma new tricks.

E2F transcription factors are thought to influence the G1-S cell-cycle transition by controlling expression of genes required for growth and DNA synthesis. But emerging evidence suggests E2F complexes can control the cell cycle independently of transcription by directly regulating DNA replication origin usage during S phase.

Acetyltransferases↗
PubMed1999

Replication origins in metazoan chromosomes: fact or fiction?

The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.

Animals↗
PubMed1987

Direction of transcription affects the replication mode of lambda in an in vitro system.

A set of artificial circular plasmids, named plasmoids, was constructed. They are about 1 kb in size and consist of a 178 bp lambda dv minimal DNA replication origin (ori) which has four direct repeats and the A + T-rich region conferring polarity to the ori fragment, a 775 bp DNA segment that codes for the CAT amino acid sequence and the 99 bp lac promoter (plac). They carry no other functional genes or genetic sites. The constructions involved various combinations of relative orientations of these components. These molecules do not replicate in vivo because they lack genes coding for initiation proteins, but they do replicate in an in vitro system (Fuller et al. 1981), and can be used for studies of interactions between transcription and replication. In these plasmids, major transcription starts from the strong plac, and some weak unscheduled transcription starts from several other initiation sites. The major RNA synthesis was found to interfere with the unscheduled RNA synthesis, which was occurring on the opposite strand. The most active replication took place when the major RNA synthesis went through the lambda origin region in the direction which occurs naturally in the lambda genome. Under these conditions, DNA synthesis going against such transcription was less than that going along with the major transcription. When RNA synthesis through the lambda origin region was in the opposite direction, DNA synthesis in the same direction was about half of that observed in the above case, whereas that going against transcription was very weak.

Bacteriophage lambda↗
PubMed2001

Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin-CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3). Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication. Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref. 10). Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.

Cdh1 Proteins↗
PubMed2001

Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates latent-phase DNA replication by an unknown mechanism that involves binding to four recognition sites in the dyad symmetry (DS) element of the viral latent origin of DNA replication. Since EBV episomes are assembled into nucleosomes, we have examined the ability of Epstein-Barr virus nuclear antigen 1 (EBNA1) to interact with the DS element when it is assembled into a nucleosome core particle. EBNA1 bound to its recognition sites within this nucleosome, forming a ternary complex, and displaced the histone octamer upon competitor DNA challenge. The DNA binding and dimerization region of EBNA1 was sufficient for nucleosome binding and destabilization. Although EBNA1 was able to bind to nucleosomes containing two recognition sites from the DS element positioned at the edge of the nucleosome, nucleosome destabilization was only observed when all four sites of the DS element were present. Our results indicate that the presence of a nucleosome at the viral origin will not prevent EBNA1 binding to its recognition sites. In addition, since four EBNA1 recognition sites are required for both nucleosome destabilization and efficient origin activation, our findings also suggest that nucleosome destabilization by EBNA1 is important for origin activation.

Animals↗