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At least 559 records · Page 31Linked to original sources
PubMed1996

A DNA structure is required for geminivirus replication origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.

Base Sequence↗
PubMed1981

A computer method for construction of secondary structure from polynucleotide sequence. Possible structure of the bacterial replication origin.

A computer method to search the possible secondary structure of a long polynucleotide was developed. As a criterion for the stabilization of a secondary structure, free energy originating from base-pairing was employed, since the structure in solution would be at the free energy minimum. The method is summarized as follows: all possible helices are collected from a given nucleotide sequence under restrictions that the length of a helix is greater than N0 bases (e.g., four bases) and the free energy of the helix calculated according to free energies of two successive sequence-dependent basepairs is lower than E0 (e.g., -5 kcal/mol). The search of secondary structures of low free energy is performed by connecting one helix to another without allowing any base-pairing between loops. For connecting single-stranded regions, destabilizing free energy of 2--3 kcal/mol is added. The method was first applied to several tRNAs and the clover-leaf structure of tRNA was obtained as a free energy minimum. Then, possible secondary structures of the replication origin regions of the Escherichia coli and Salmonella typhimurium chromosomes were examined by the method, assuming that one of the strands in the origin region takes a specific secondary structure. The lowest-energy structure for the E. coli origin was found to be approximately identical to that for the S. typhimurium origin region.

Base Sequence↗
PubMed1983

Reciprocal and nonreciprocal recombination in diploid clones from Bacillus subtilis protoplast fusion: Association with the replication origin and terminus.

The primary heterodiploid bacteria regenerated after Bacillus subtilis fusion, although generally noncomplementing diploids, behave in pedigree analysis as multipotential systems. Individual diploid colonies yielding complete reciprocal recombinant (RR) progeny-often accompanied by one or both parents-constitute 10-30% of the total recombinant-forming units. The RR (reciprocal for 8-11 genes) usually occur in equivalent numbers both among and within individual colonies. Novel for bacteria, they demonstrate that entire parental genomes brought together within a diploid protoplast are retained as two independent replicons able to undergo classical recombination characteristic of eukaryotic gametogenesis. Parental or recombinant genomes are also subject to multiple rounds of recombination without obligate segregation and often not reciprocal. Diploid recombinant clones, sharing streptomycin resistance but reciprocal for auxotrophic markers, have displayed a partial ability to make a facultative shift in chromosome expression. They have also produced two types of prototrophs: a stable one (presumably haploid and recombinant) and an unstable one, (diploid and temporarily complementing at low frequency). It follows that chromosome extinction may affect both parental and recombinant chromosomes and does not interfere with recombination. Analysis of the number and chromosomal distribution of crossovers in all recombinants and those from single diploid clones shows increased frequency of exchange in the regions of the replication origin and terminus, possibly a result of the association of these sites with the cell wall or membrane.

Journal Article↗
PubMed1995

Replication enhancer requirement for recognition of heterologous replication origin by an initiator protein.

In the pT181 plasmid family, the replication initiation protein (Rep) encoded by each plasmid recognizes only its cognate origin, unless the Rep protein is expressed at abnormally high levels. Heterologous recognition of the origin of the pC221 plasmid by the RepC protein of the pT181 plasmid requires that cmp, the pT181 replication enhancer, be present on the same plasmid as the origin of replication. These findings indicate that cmp has a role in the specificity of Rep-ori recognition and support the model that cmp facilitates the formation/stabilization of the RepC-origin complex.

Bacterial Proteins↗
PubMed2003

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

DNA↗
PubMed1989

DNA replication origin and transcriptional enhancer in c-myc gene share the c-myc protein binding sequences.

We have previously reported that c-myc protein, or protein(s) complexed with c-myc protein, binds to the region upstream of the first exon of the c-myc gene and that this region contains an origin of cellular DNA replication (ori) and also a transcriptional enhancer. Here we show by Southwestern blotting that c-myc protein binds directly to a 7 bp sequence within the above region. Furthermore, we show that the c-myc protein binding sequences are indispensable for both ori and enhancer functions, but that additional sequences are required for maximal ori and enhancer activities. Thus, c-myc protein is a sequence specific factor which is apparently used both in initiation of DNA replication and in regulation of RNA transcription.

Animals↗
PubMed1981

Mutation near the polyoma DNA replication origin permits productive infection of F9 embryonal carcinoma cells.

F9 mouse embryonal carcinoma cells are resistant to productive infection by wild-type polyoma virus. Continued passage of F9 cells initially infected with wild-type polyoma virus eventually leads to the selection of polyoma virus mutants that are capable of productive infection of undifferentiated F9 cells. Three mutants, PyF101, PyF111 and PyF441, have been plaque-purified and examined. All three PyF mutant DNAs are altered from the wild-type sequence in the Pvu II-4 fragment that spans 67.6 to 70.2 map units on the polyoma genome. PyF441 has a single base change of A to G at 69.6 map units. PyF101 and PyF111 DNAs also contain this point mutation at 69.6 map units. In addition, PyF101 and PyF111 DNAs have exact tandem duplications of 54 and 31 bp, respectively, of sequences encompassing the point mutation, and both copies of the tandem duplication have the point mutation. Other than these changes, no difference exists in the nucleotide sequences of wild-type and PyF mutant DNAs from the BcI I site at 65.6 map units clockwise through the origin of viral DNA replication to the BgI I site at 72.2 map units. DNA infections of F9 cells with wild-type-mutant hybrid DNAs formed by ligation of heterologous combinations of the small and large DNA fragments generated by double digestion with the restriction enzymes BcII and BGI I show that the DNA sequence changes described above are responsible for the ability of the PyF mutants to infect F9 cells.

Animals↗
PubMed1990

Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene.

The c-myc genes of HeLa cells are preferentially replicated in the transcriptional direction, from chromosomal origin sequences which display cell type-specific activity. Using a run-off replication assay involving in vitro extension of replication forks initiated in intact HeLa cells, bidirectional replication was observed to begin within a 3.5 kb domain 5' to the c-myc gene. To characterize the replication origin further a 2.4 HindIII-Xhol subfragment of the c-myc 5' flanking DNA was cloned in a selectable vector and transfected into HeLa cells. The resulting pNeo.Myc-2.4 construct persisted as a circular extrachromosomal element for more than 300 cell generations under selection, with recovery of approximately 500-1000 times the mass of plasmid initially introduced into the cells. Extrachromosomal circular pNeo.Myc-2.4 monomer was reisolated in supercoiled form, along with oligomeric and miniplasmid variants which had been generated in vivo; however, chromosomally integrated copies of the plasmid were not detectable in cultures containing extrachromosomal pNeo.Myc-2.4. The recovered pNeo.Myc-2.4 plasmid was resistant to Dpnl digestion and sensitive to Mbol digestion. After transfection with pNeo.Myc-2.4 BrUdR pulse labeling of long-term or short-term cultures demonstrated that the plasmid replicated semiconservatively, under controls similar to those imposed on chromosome replication. Bisection of the pNeo.Myc-2.4 insert suggested that c-myc 5' flanking DNA within 1.2 kb 5' to promoter P1 was sufficient to confer autonomously replicating sequence activity on the plasmid vector in transient replication assays.

Chromosomes, Human↗
PubMed1992

Sequence analysis of replication origin of plasmid pLS11 of Bacillus subtilis IFO 3022.

The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS11 of Bacillus subtilis IFO 3022, which is responsible for gamma-polyglutamate production, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. subtilis TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8% identity), pBAA1 (92.8%), and pFTB14 (78.7%) in Bacillus spp., pLP1 (42.1%) and pLAB1000 (36.3%) in Lactobacillus spp., and pUB110 (35.3%) and pC194 (37.4%) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.

Amino Acid Sequence↗
PubMed1996

Identification of a putative DNA replication origin in the gamma-aminobutyric acid receptor subunit beta3 and alpha5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing.

The region containing the GABAA receptor beta3 and alpha5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone lambda84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage lambda84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13kb of contiguous sequence reveals several A + T-rich regions which display potential DNA unwinding. The plasmid subclones from phage lambda84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABAA receptor subunit gene cluster.

Alleles↗
PubMed1996

A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis.

Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate. Encapsidation of this RNA pregenome is mediated by the interaction of the viral replication enzyme P with the structured 5'-proximal RNA element epsilon; replication was thought to start in the 3'-proximal direct repeat DR1*. However, recent data obtained with the duck hepatitis B virus indicated a novel, discontinuous mechanism of negative-strand DNA synthesis. Here we demonstrate, using DNA transfection of complete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV epsilon whose primary sequence is not important for encapsidation serves as template for a short DNA primer that is subsequently transferred to DR1*. Apparently, P protein copies any template sequence that does not interfere with epsilon structure; however, altered primary sequences can induce polymerase stuttering, resulting in extended primers containing more than one equivalent of the template sequence. The importance of the bulged structure is emphasized by the dependence of primer length on bulge size. Transfer specificity is in part controlled by sequence complementarity. The strategy of using the 5' encapsidation signal as the origin of replication for discontinuous negative-strand DNA synthesis, common to mammalian and avian hepadnaviruses, suggests the evolutionary origin of hepatitis B viruses to lie between that of modern retroviruses and primitive retroelements like the Mauriceville retroplasmid.

Base Sequence↗
PubMed1980

Activity in vitro of three replication origins of the antibiotic resistance plasmid RSF1040.

Replicating molecules of plasmid RSF1040, a deletion mutant of R6K, were synthesized in vitro and analyzed by electron microscopy. Initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma, within a 3900-base pair segment of the R6K genome. These sites are indistinguishable from the origins that are active in vivo. Frequencies of initiation at these three origins, however, are different from those observed in vivo. Replication proceeds unidirectionally in either direction from ori beta and ori gamma and in one direction from ori alpha. The replication terminus of the R6K genome is inactive in the in vitro system.

Anti-Bacterial Agents↗
PubMed1986

Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii.

Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5' end and 0.43 kb to the 3' end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.

Journal Article↗
PubMed1977

Physical structure of the replication origin of bacteriophage lambda.

The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined. Three of the ori- mutations are small deletions, and one is a transversion. The sequence of the origin region, defined by these mutations, contains a number of unusual features.

Base Sequence↗
PubMed1993

cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae.

The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication. We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence. Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential. A perfect match to a revised ARS consensus is present but not essential. Sequences up to 9 bp 5' from the essential consensus are dispensable. A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence. A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element. Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element. The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.

Base Sequence↗
PubMed1987

ATP-dependent formation of a specialized nucleoprotein structure by simian virus 40 (SV40) large tumor antigen at the SV40 replication origin.

The large tumor antigen (T antigen) specified by simian virus 40 (SV40) is required for viral DNA replication. To carry out its function, T antigen binds to duplex DNA at the origin of replication (oriSV40) and exerts a helicase activity that unwinds the two DNA strands. Previous work has defined two binding sites for T antigen near oriSV40, designated sites I and II; site II is within the 64-base-pair core sequence absolutely required for viral DNA replication. We have used electron microscopy and gel electrophoresis to characterize the interaction of T antigen with the origin region. We have found that effective binding to site II under conditions that support DNA replication requires ATP or a nonhydrolyzable analog. In the absence of ATP, T antigen binds mainly to site I; in the presence of ATP, both sites I and II are occupied, and binding is markedly increased. The ATP-dependent reaction generates a complex multimeric structure for T antigen. We conclude that T antigen forms an ATP-dependent nucleoprotein structure at oriSV40. We suggest that this nucleoprotein complex provides for the precise initiation of SV40 DNA replication.

Adenosine Triphosphate↗
PubMed2001

Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer.

BACKGROUND: Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized. RESULTS: Systematic deletion of consecutive segments of approximately 50, approximately 100 or approximately 150 bp within the enhancer and its adjacent core origin (ars3002) revealed that several of the approximately 50-bp stretches within the enhancer contribute to its function in partially redundant fashion. Other stretches within the enhancer are inhibitory. Some of the stretches within the enhancer proved to be redundant with sequences within core ars3002. Consequently the collection of sequences important for core origin function was found to depend on whether the core origin is assayed in the presence or absence of the enhancer. Some of the important sequences in the core origin and enhancer co-localize with short runs of adenines or thymines, which may serve as binding sites for the fission yeast Origin Recognition Complex (ORC). Others co-localize with matches to consensus sequences commonly found in fission yeast replication origins. CONCLUSIONS: The enhancer within the ura4 origin cluster in fission yeast contains multiple sequence motifs. Many of these stimulate origin function in partially redundant fashion. Some of them resemble motifs also found in core origins. The next step is to identify the proteins that bind to these stimulatory sequences.

Journal Article↗
PubMed1987

The in vivo replication origin of the yeast 2 microns plasmid.

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.

DNA Replication↗