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At least 505 records · Page 28Linked to original sources
PubMed1991

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication.

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.

Adenoviridae↗
PubMed1989

Binding of the herpes simplex virus type 1 UL9 gene product to an origin of viral DNA replication.

The binding of a herpes simplex virus type 1 (HSV-1) encoded polypeptide to a viral origin of DNA replication has been studied by using a gel retardation assay. Incubation of nuclear extract from HSV-1 infected cells with a labelled origin-containing fragment resulted in the formation of a specific retarded complex, the migration of which was further reduced in the presence of an antibody reactive with the UL9 gene product. Introduction of an additional copy of the UL9 gene, under the control of an immediate early (IE) promoter, conferred the ability to express origin binding activity at the non-permissive temperature upon an HSV-1 ts mutant blocked at the IE stage of infection. Endogenous or exogenous proteolytic activity revealed the presence of a relatively protease-resistant domain which retained sequence-specific DNA binding activity. The C-terminal 317 amino acids of the UL9 gene expressed as a fusion protein in Escherichia coli also bound to the origin. Our results demonstrate that the UL9 gene product binds to a viral origin and that sequence specific recognition and binding are specified by the C-terminal 37% of the polypeptide.

Animals↗
PubMed1995

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.

Animals↗
PubMed1996

Transcriptional terminators of RNA polymerase II are associated with yeast replication origins.

The compact organization of the Saccharomyces cerevisiae genome necessitates that non-coding regulatory sequences reside in close proximity to one another. Here we show there is an intimate association between transcription terminators and DNA replication origins. Four replication origins were analyzed in a reporter gene assay that detects sequences that direct 3' end formation of mRNA transcripts. All four replication origins function as orientation-independent transcription terminators in this system, producing truncated polyadenylated mRNAs. Despite this close association, the cis-acting elements that confer replication origin function are genetically separable from those required for transcription termination. Several models are explored in an attempt to address how and why the signals specifying transcription termination and replication initiation overlap.

Base Sequence↗
PubMed2001

RNA polymerase II and III transcription factors can stimulate DNA replication by modifying origin chromatin structures.

Many transcription factors are multifunctional and also influence DNA replication. So far, their mechanism of action has remained elusive. Here we show that a DNA-binding protein could rely on the same biochemical activity that activates transcription to stimulate replication from the yeast chromosomal ARS1 origin. Unexpectedly, the ability to stimulate replication from this origin was not restricted to polymerase II transcription factors, but was a property shared by polymerase III factors. Furthermore, activation of replication did not depend on the process of transcription, but rather on the ability of DNA-binding transcription factors to remodel chromatin. The natural ARS1 activator Abf1 and the other transcription factors that stimulated replication remodeled chromatin in a very similar manner. Moreover, the presence of a histone H3 mutant that was previously shown to generally increase transcription also facilitated replication from ARS1 and partially compensated for the absence of a transcription factor. We propose that multifunctional transcription factors work by influencing the chromatin architecture at replication origins so as to generate a structure that is favorable to the initiation of replication.

Binding Sites↗
PubMed2003

In vitro protein-DNA interactions at the human lamin B2 replication origin.

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition.

Adenosine Triphosphate↗
PubMed1994

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA.

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.

Animals↗
PubMed1995

A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence.

Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5' end of the rG region. The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required. The efficiency of hybrid formation depends on the length of RNA synthesized 5' to CSBII and the type of RNA polymerase employed. Once made, the RNA strand of an RNA-DNA hybrid can serve as an effective primer for mitochondrial DNA polymerase. These results reveal a new mechanism for persistent RNA-DNA hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA hybrid.

Base Sequence↗
PubMed1997

Mammalian cell DNA replication.

The precise mechanisms involved in the regulation of the mammalian cell DNA-synthesizing machinery are poorly understood. In vitro DNA replication systems, in particular the employment of the simian virus 40 (SV40)-based cell-free DNA replication system, has identified several mammalian enzymes and proteins required for DNA synthesis. Although these proteins have been identified as playing a role in DNA replication, their functional organization allowing for the efficient replication of DNA has not been well defined. This review describes the proteins that have currently been defined as having a role in mammalian DNA replication and their proposed mechanisms of action. How these proteins may organize themselves to form multiprotein complexes, or larger DNA replication factories, allowing for efficient chromosomal DNA synthesis is discussed. In addition, the cell cycle regulation of mammalian DNA synthesis and the current status concerning mammalian DNA replication origins is described.

Animals↗
PubMed1988

Deletion analysis of bacteriophage T4 tertiary origins. A promoter sequence is required for a rifampicin-resistant replication origin.

The DNA sequence requirements of two T4 tertiary replication origins have been characterized by a deletion analysis of origin-containing plasmids. Maximal replication of each origin-containing plasmid required both an intact gpmotA-dependent middle-mode promoter sequence and approximately 50 base pairs of the downstream region. In contrast, gpmotA-dependent transcription from the origin promoter was found to be independent of the downstream region. The requirement for a promoter element within the tertiary origins is striking, particularly since the replication of tertiary origin-containing plasmids is resistant to the RNA polymerase inhibitor rifampicin.

Base Sequence↗
PubMed1996

Cdc6 and DNA replication: limited to humble origins.

The budding yeast Cdc6 protein is important for regulating DNA replication initiation. Cdc6p acts at replication origins, and cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G1, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.

Cell Cycle↗
PubMed1988

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.

Chloroplasts↗
PubMed2004

Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.

A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (approximately 36 kb) at the 5' end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.

Animals↗
PubMed1994

Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo.

DNA replication in the budding yeast Saccharomyces cerevisiae initiates from origins of specific DNA sequences during S phase. A screen based on two- and one-hybrid approaches demonstrates that the product of the DBF4 gene interacts with yeast replication origins in vivo. The Dbf4 protein interacts with and positively regulates the activity of the Cdc7 protein kinase, which is required for entry into S phase in the yeast mitotic cell cycle. The analysis described here suggests a model in which one function of Dbf4 may be to recruit the Cdc7 protein kinase to initiation complexes.

Base Sequence↗
PubMed1992

Eukaryotic replication origins as promoters of bidirectional DNA synthesis.

Recent work in yeast shows that eukaryotic origins of DNA replication are multipartite regulatory elements resembling promoters of transcription. As for the regulation of transcription, accessory transcription factors appear to function in concert with basic origin recognition factors to regulate initiation of DNA synthesis at specific subsets of origins. The participation of transcription factors in the regulation of DNA replication may facilitate temporal control of transcription and replication during the cell cycle, as well as providing a mechanism for integrating origin selection with the cellular transcriptional program.

DNA Mutational Analysis↗
PubMed1998

Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis.

We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1). The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.

Bacillus subtilis↗
PubMed1985

Identification of the short dispersed repetitive DNA sequences isolated from the zones of initiation of DNA synthesis in human cells as Alu-elements.

DNA of Xeroderma pigmentosum cells was crosslinked in vivo with trioxsalen and long wave length ultraviolet light and the cells were cultured in the presence of labelled thymidine for one hour. The nascent DNA chains synthesized during this period and containing the DNA replication origins were isolated from the high molecular weight chromosomal DNA by an alkaline sucrose density gradient centrifugation. They were 5-10-fold enriched in short dispersed repetitive sequences identified by dot-blot hybridization to BLUR 8 plasmid as members of the human Alu-family.

DNA↗
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↗