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At least 1,225 records · Page 68Linked to original sources
PubMed1995

The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators.

Replicators are genetically defined elements within chromosomes that determine the location of origins of DNA replication. In the yeast Saccharomyces cerevisiae, the ARS1 replicator contains multiple functional DNA elements: an essential A element and three important B elements--B1, B2, and B3. Functionally similar A, B1, and B2 elements are also present in the ARS307 replicator. The B3 element binds a replication and transcription enhancer protein Abf1p, whereas the A element is required for binding the origin recognition complex (ORC). The function of the B1 and B2 elements remains to be defined. We have used a gel-based DNA binding assay to study the interaction between replicators and the putative initiator protein ORC. In addition to the established requirements for ATP and the A element for ORC-DNA interaction, the new data demonstrate that sequences in the B1 element are also important for ORC-DNA association. This conclusion is supported by DNase I footprint analyses and demonstrates that ORC binds to a bipartitite recognition element within the DNA. Furthermore, mutation of nucleotides in the B1 element suggests that this element has other functions in the initiation of DNA replication besides participating in the ORC-DNA interaction.

Base Sequence↗
PubMed1994

Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori- plasmids and their in vivo chromosomal integration and retrieval.

We have constructed a plasmid system designed for the insertion of cloned DNA (e.g., genes, gene fusions, regulatory elements, etc.) into the Escherichia coli genome. Its principal feature is the presence of two tandem lox sites on the plasmids, which upon Cre-mediated in vitro recombination resolve the plasmids into ori- and ori+ DNA circles. The non-replicating ori- circles contain the lambda attP site, several unique restriction sites for cloning, a NotI site and KmR, a kanamycin-resistance-encoding gene. The ori+ circles carry the origin of DNA replication (ori) together with several cleavage sites not present in the ori- circles, including the rare site for the very efficient I-SceI enzyme, that are used to inactivate the ori+ circles and any unresolved plasmid DNA. We have used this system to insert cloned DNA into the host genome at (i) the attB site, by Int-mediated integration and (ii) at any predetermined sequence, as mediated by the Rec system(s) of the host. The genomes of the resulting transformants were analyzed by NotI digestion of the chromosomal DNA, embedded in agarose microbeads, followed by pulsed-field gel electrophoresis. A system for the retrieval of DNA fragments inserted at the attB site was also developed.

Base Sequence↗
PubMed2006

Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks.

The Cdc45 protein is crucial for the initiation of chromosome replication in eukaryotic cells, as it allows the activation of prereplication complexes (pre-RCs) that contain the MCM helicase. This causes the unwinding of origins and the establishment of DNA replication forks. The incorporation of Cdc45 at nascent forks is a highly regulated and poorly understood process that requires, in budding yeast, the Sld3 protein and the GINS complex. Previous studies suggested that Sld3 is also important for the progression of DNA replication forks after the initiation step, as are Cdc45 and GINS. In contrast, we show here that Sld3 does not move with DNA replication forks and only associates with MCM in an unstable manner before initiation. After the establishment of DNA replication forks from early origins, Sld3 is no longer essential for the completion of chromosome replication. Unlike Sld3, GINS is not required for the initial recruitment of Cdc45 to origins and instead is necessary for stable engagement of Cdc45 with the nascent replisome. Like Cdc45, GINS then associates stably with MCM during S-phase.

Cell Cycle Proteins↗
PubMed2024

Strategic targeting of Cas9 nickase induces large segmental duplications.

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.

Saccharomyces cerevisiae↗
PubMed1981

A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.

Animals↗
PubMed1994

The role of a pentanucleotide repeat sequence, AGGGAAGGGA, in the regulation of JC virus DNA replication.

The human polyomavirus JCV differs from other papovaviruses in its narrow host range and tissue tropism for human glial cells. It is believed that the cell-specific tropism of JCV to glial cells rests, at least in part, in transcription of the viral early gene that encodes the large tumor antigen (T-antigen). The secondary stage, however, which restricts the replication cycle of JCV to primate cells, is controlled at the level of viral DNA replication. In this study, we demonstrate that a cis-acting transcription regulatory element encompassing the pentanucleotide repeat sequence AGGGAAGGGA (penta), which is located in close proximity to the origin of DNA replication, plays an important role in the replication of viral DNA mediated by the JCV T-antigen, but not T-antigen derived from SV40. Analysis of DNA structure by diethyl pyrocarbonate (DEPC) has revealed that mutations within the penta which affect DNA replication also alter the structure of the neighboring A+T-rich region. These results suggest that, in addition to the regulatory role in viral gene expression, the penta may function as a DNA structural element which is important for JCV DNA replication mediated by the JCV T-antigen.

Animals↗
PubMed1994

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.

Animals↗
PubMed2006

Is there a pre-RC checkpoint that cancer cells lack?

Critical for genomic integrity, accurate DNA replication is tightly regulated by the convergence of prereplication protein complexes (pre-RCs) to "license" replicating origins on DNA in G(1) and is activated by S-phase promoting kinases that selectively target and trigger origin firing in S-phase. To present, a checkpoint mechanism monitoring pre-RC complex formation and activation has yet to be elucidated. However, perturbation of these protein complexes has yielded divergent phenotypes in recent reports: normal cells arrest in the cell cycle, whereas cancerous cells arrest and die. These data implicate a mechanism by which normal cells sense pre-RC deficiency and then signal for cell cycle arrest. The potential for therapeutic exploits of this disparity between normal and cancer cells is apparent. Here, we explore recent data supporting the existence of a pre-RC checkpoint that ensures faithful pre-RC formation, a cell cycle mechanism that is intriguingly compromised in cancer cells.

Animals↗
PubMed1992

Epstein-Barr virus DNA recombines via latent origin of replication with the human genome in the lymphoblastoid cell line RGN1.

We show here that in a lymphoblastoid cell line Epstein-Barr virus DNA recombines with the human genome. The genetic exchange involves the oriP region of the virus. A junction between viral and human DNA from this line has been cloned and sequenced. The results indicate that the integration of Epstein-Barr virus DNA involves a region of the human genome which contains internal short repetition. An 800-bp probe has been isolated from the human part of the junction. This probe has been used to show that the human region exists as a duplication in normal cells.

Base Sequence↗
PubMed1997

Cyclin-dependent kinase regulation of the replication functions of polyomavirus large T antigen.

The amino-terminal portion of polyomavirus (Py) large T antigen (T Ag) contains two phosphorylation sites, at T187 and T278, which are potential substrates for cyclin-dependent kinases (CDKs). Our experiments were designed to test whether either or both of these sites are involved in the origin DNA (ori DNA) replication function of Py T Ag. Mutations were generated in Py T Ag whereby either or both threonines were replaced with alanine, generating T187A, T278A, and double-mutants (DM [T187A T278A]) mutant T Ags. We found that the Py ori DNA replication functions of T278A and DM, but not T187A, mutant T Ags were abolished both in vivo and in vitro. Consistent with this finding, it was shown that the ori DNA binding and unwinding activities of mutant T278A Py T Ag were greatly impaired. Moreover, whereas wild-type Py T Ag is an efficient substrate for phosphorylation by cyclin A-CDK2 and cyclin B-cdc2 complexes, it is phosphorylated poorly by a cyclin E-CDK2 complex. In contrast to mutant T187A, which behaved similarly to the wild-type protein, T278A was only weakly phosphorylated by cyclin B-cdc2. These data thus suggest that T278 is an important site on Py T Ag for phosphorylation by CDKs and that loss of this site leads to its various defects in mediating ori DNA replication. S- and G2-phase-specific CDKs, but not a G1-specific CDK, can phosphorylate wild-type T Ag, which suggests yet another reason why DNA tumor viruses require actively cycling host cells.

Antigens, Polyomavirus Transforming↗
PubMed2006

In vitro and in vivo stability of plasmids in attenuated Salmonella enterica serovar typhimurium used as a carrier of DNA vaccine is associated with its replication origin.

The ability of live attenuated Salmonella enterica serovar typhimurium (S. typhimurium) as a carrier of DNA vaccine was evaluated using model plasmid encoding beta-galactosidase (beta-Gal) and BALB/c mice. We constructed pBRCMVbeta, beta-Gal expression apparatus having a replication origin from low copy pBR322. Comparison of the plasmid stability showed that pBRCMVbeta remained stable in Salmonella even after oral administration, while pUC-based pCMVbeta tended to be lost quickly. However, titers for beta-Gal specific IgG in sera did not significantly increase in mice orally administered S. typhimurium harboring pBRCMVbeta. These data suggest that the stability of plasmid in S. typhimurium is associated with its replication origin. Further studies are required to scientifically establish this methodology.

Administration, Oral↗
PubMed1992

In vivo duplication of genetic elements by the formation of stem-loop DNA without an RNA intermediate.

Gene duplication through cDNA synthesis by reverse transcriptase is believed to have played an important role in the diversification of genomes during evolution. Here, we demonstrate that a genomic DNA sequence can be duplicated in vivo as a result of template switching. When an inverted repeat (IR) structure was inserted in a site downstream from a ColE1 plasmid origin of DNA replication, transformation of Escherichia coli cells with this plasmid resulted in the production of a new DNA fragment encompassing the region from the origin to the center of the IR structure. The structure of this DNA molecule is composed of a long stem-loop formed by a single-stranded DNA, in which the loop is formed by the IR structure. The DNA fragment is designated slDNA, for stem-loop DNA. The experiments in this study suggest that during DNA replication, template switching at the stem-loop structure formed by the IR structure gives rise to slDNA utilizing the nascent DNA strand or the parental strand as a template. The mechanistic implications of slDNA synthesis, and its possible roles in genome evolution, are discussed.

Base Sequence↗
PubMed1985

DNA sequence of the region in the genome of herpes simplex virus type 1 containing the genes for DNA polymerase and the major DNA binding protein.

In the long unique region of the genome of herpes simplex virus type 1 (HSV-1), the genes for DNA polymerase and the major DNA binding protein are arranged in a head to head manner, with an origin of DNA replication (termed OriL) located between them. This paper reports an 8400 base pair DNA sequence containing both genes and the origin, obtained mostly by M13/dideoxy analysis of plasmid cloned fragments. Amino acid sequences of the two proteins were deduced. Homologues of both genes were detected in the genome sequence of the distantly related Epstein-Barr virus (EBV). Arrangement of these HSV-1 and EBV genes differs in genome location and in relative orientation. A part of HSV-1 DNA polymerase was found to be similar to a sequence in adenovirus 2 DNA polymerase, but the significance of this is unclear. Since a DNA sequence in the locality of OriL deletes on plasmid cloning, this region was analysed using virus DNA. A palindrome with 72-residue arms was found, which shows great similarity to the better characterized origin, OriS.

Adenoviridae↗
PubMed2001

Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis.

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.

Bacterial Proteins↗
PubMed1995

Supercoiling and map stability in the bacterial chromosome.

A major goal of comparative genomics is an understanding of the forces which control gene order. This assumes that gene order is important, a supposition backed by the existence of genomic colinearity between many related species. In the bacterial chromosome, a polarity in the order of genes has been suggested, influenced by distance and orientation relative to the origin of DNA replication. We propose a model of the bacterial chromosome in which gene order is maintained by the adaptation of gene expression to local superhelical context. This force acts not directly at the genomic level but rather at the local gene level. A full understanding of gene-order conservation must therefore come from the bottom up.

Biological Evolution↗
PubMed1992

The region essential for efficient autonomous replication of pSa in Escherichia coli.

Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.

Amino Acid Sequence↗
PubMed1992

Transient replication of human papillomavirus DNAs.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.

Base Sequence↗