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At least 865 records · Page 48Linked to original sources
PubMed2003

EBNA1 efficiently assembles on chromatin containing the Epstein-Barr virus latent origin of replication.

The Epstein-Barr virus (EBV) protein, EBNA1, activates the replication of latent EBV episomes and the transcription of EBV latency genes by binding to recognition sites in the DS and FR elements of oriP. Since EBV episomes exist as chromatin, we have examined the interaction of EBNA1 with oriP templates assembled with physiologically spaced nucleosomes. We show that EBNA1 retains the ability to efficiently bind its recognition sites within the DS and FR elements in oriP chromatin and that this property is intrinsic to the EBNA1 DNA binding domain. The efficient assembly of EBNA1 on oriP chromatin does not require ATP-dependent chromatin remodeling factors and does not cause the precise positioning of nucleosomes within or adjacent to the FR and DS elements. Thus EBNA1 belongs to a select group of proteins that can efficiently access their recognition sites within nucleosomes without the need for additional chromatin remodeling factors.

Chromatin↗
PubMed1995

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

Base Sequence↗
PubMed1999

A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells.

We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure.

Animals↗
PubMed1989

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.

Base Sequence↗
PubMed1981

DNA sequences similar to those around the simian virus 40 origin of replication are present in the monkey genome.

We report the molecular cloning of African green monkey genomic DNA segments that include regions of homology to the origin of replication of simian virus 40 (SV40). Three clearly different cloned segments 14 to 17 kilobase pairs (kb) long were isolated from a genomic library in lambda phage. We estimate that each of the three is repeated fewer than four times in the monkey genome. The SV40-like regions represent a small portion of the cloned segments, and these regions cross hybridize only weakly with one another. One of the three segments is described here in detail. Although the entire segment occurs only once or twice in the monkey genome, it contains DNA sequences (other than the SV40-like sequences) that are repeated elsewhere in the genome including in the other two cloned segments. The homology to SV40 is contained within about 300 base pairs of monkey DNA and is limited to the region around the viral replication origin. The nucleotide sequence of the SV40-like region was determined. It contains a large number of short stretches homologous to three specific noncoding domains around the SV40 origin of replication: the 27-base-pair region of dyad symmetry, the first set of (short) repeats that occur just on the late side of the origin, and, further in the late direction, the two 72-base-pair-long repeats. Although these components are grouped in the monkey DNA, as they are in SV40 DNA, their relative juxtaposition is scrambled.

Animals↗
PubMed1991

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.

Animals↗
PubMed2006

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid.

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.

Bacterial Proteins↗
PubMed2005

CDK-dependent stabilization of Cdc6: linking growth and stress signals to activation of DNA replication.

Cyclin-dependent kinases (CDKs) play a crucial role in cell cycle progression by controlling the transition from G(1) phase into S phase where DNA is replicated. Key to this transition is the regulation of initiation of DNA replication at replication origins. CDKs are thought to regulate origins of replication both positively and negatively by phosphorylating replication proteins at origins. Several replication proteins that are potentially negatively regulated upon CDK phosphorylation have been identified. However, the mechanism by which CDKs activate replication is currently less well understood. New observations revealing that the initiation protein Cdc6 is stabilized by CDK2-dependent phosphorylation may give more insight in this process.

Cell Cycle Proteins↗
PubMed1984

Monkey B-lymphotropic papovavirus DNA: nucleotide sequence of the region around the origin of replication.

We have determined the nucleotide sequence of the HindIII-B DNA segment of African green monkey B-lymphotropic papovavirus (LPV), which shows a highly restricted host range and whose genome is a 5.1-kilobase-long circular DNA. The segment, consisting of 1,123 base pairs, contained the origin of DNA replication, the putative control region for early transcription, and the region probably coding for the amino-terminal portion of T antigens. The symmetrical region at the center of replication origin, 5'-GAGGC CA GGGGCCCC TA GCCTC-3' (on the L strand), has diverged in the central portion from the corresponding regions of primate polyomaviruses simian virus 40, BK virus, and JC virus, but resembles that of mouse polyomavirus. The structure of the control region upstream of the replication origin was unique to LPV and contained several repeated sequences, the longest of which were two 60-base-pair tandem repeats. The amino-terminal region common to LPV small T and large T antigens showed some homology (41%) in the deduced amino acid sequence to that of both simian virus 40 and the mouse polyomavirus. Like other polyomaviruses, the probable carboxyl-terminal region unique to LPV small T antigen contained two sets of the Cys-x-Cys-x-x-Cys structure. These data show that, despite the unique structures in the control region, LPV is evolutionally related to the mouse polyomavirus and to simian virus 40.

Amino Acid Sequence↗
PubMed2000

Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei.

Adjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication. Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase. Orf770 showed similarity to putative plasmid and phage DNA primases. Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection. When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation. It is deduced that this stretch contains the origin of replication of phage A2.

Amino Acid Sequence↗
PubMed1981

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2.

A DNA sequence consisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc2 incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc2 incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G + C content is present. Deletion evidence indicates that the A-T rich and possibly the G + C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.

Base Sequence↗
PubMed1999

Influence of a replication enhancer on the hierarchy of origin efficiencies within a cluster of DNA replication origins.

DNA replication origins in animal cells sometimes occur in clusters. Often one of the multiple origins within these clusters fires more frequently than the others. The reason for this hierarchy remains unknown. Similar origin clusters occur in the fission yeast, Schizosaccharomyces pombe. One such cluster is located near the ura4 gene on chromosome III and contains three origins: ars3002, ars3003, and ars3004. In their natural chromosomal context (ars3003 is about 2.5 kb upstream of ars3002 and ars3004 is adjacent to ars3002 on the downstream side) their initiation frequencies display a striking hierarchy: ars3002 >> ars3003 >> ars3004. Here, we describe experiments that reveal a 400 bp replication enhancer within ars3004, adjacent to ars3002. The enhancer is essential for ars3004 origin function in a plasmid, but even with the enhancer ars3004 is an inefficient origin. The enhancer is not essential for ars3002 plasmid origin activity, but dramatically stimulates this activity, converting ars3002 from an inefficient plasmid origin to a very efficient one. It also stimulates the plasmid origin activity of ars3001 and ars3003 at all tested positions and orientations on both sides of each autonomously replicating sequence (ARS) element. If ars3002 is redefined to include the enhancer, then the relative activities of the three ARS elements as single origins within separate plasmids or as origins when all three ARS elements are present in a single plasmid is the same as the chromosomal hierarchy. Thus, this replication enhancer defines the relative activities of the three origins in the ura4 origin region. Similar enhancers may affect relative activities in the origin clusters of animal cells.

Cell Count↗