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PubMed1988

An initiation site of DNA replication with transcriptional enhancer activity present upstream of the c-myc gene.

We have previously reported that c-myc protein may promote cellular DNA replication by binding to initiation sites of replication. Here we report that a putative origin of human cellular DNA replication (ori) is present at approximately 2 kb upstream of the coding region of the c-myc gene itself. The c-myc protein, or protein(s) complexed with c-myc protein, bind to the upstream region (approximately 200 bp in length) which has transcriptional enhancer activity as well as autonomously replicating activity in human cells, suggesting that the c-myc protein may be an enhancer binding protein as well as a DNA replication protein. Results with deletion mutants suggest that the sequence essential to the origin of DNA replication may be adjacent to, but cannot be clearly separated from, the sequence responsible for enhancer activity. Furthermore, when cloned DNA containing putative c-myc protein binding sequences was transfected as competitor into HL-60 cells, expression of c-myc was inhibited, suggesting that c-myc protein itself may be necessary for c-myc expression.

Base Sequence↗
PubMed1998

The herpes simplex virus type-1 single-strand DNA-binding protein, ICP8, increases the processivity of the UL9 protein DNA helicase.

Herpes simplex virus type-1 UL9 protein is a sequence-specific DNA-binding protein that recognizes elements in the viral origins of DNA replication and possesses DNA helicase activity. It forms an essential complex with its cognate single-strand DNA-binding protein, ICP8. The DNA helicase activity of the UL9 protein is greatly stimulated as a consequence of this interaction. A complex of these two proteins is thought to be responsible for unwinding the viral origins of DNA replication. The aim of this study was to identify the mechanism by which ICP8 stimulates the translocation of the UL9 protein along DNA. The data show that the association of the UL9 protein with DNA substrate is slow and that its dissociation from the DNA substrate is fast, suggesting that it is nonprocessive. ICP8 caused maximal stimulation of DNA unwinding activity at equimolar UL9 protein concentrations, indicating that the active species is a complex that contains UL9 protein and ICP8 in 1:1 ratio. ICP8 prevented dissociation of UL9 protein from the DNA substrate, suggesting that it increases its processivity. ICP8 specifically stimulated the DNA-dependent ATPase activity of the UL9 protein with DNA cofactors that allow translocation of UL9 protein and those with secondary structure. These data suggest that UL9 protein and ICP8 form a specific complex that translocates along DNA. Within this complex, ICP8 tethers the UL9 protein to the DNA substrate, thereby preventing its dissociation, and participates directly in the assimilation and stabilization of the unwound DNA strand, thus facilitating translocation of the complex through regions of duplex DNA.

Adenosine Triphosphatases↗
PubMed1982

Origin of replication of Escherichia coli plasmid RSF 1030.

The nucleotide sequence of a region of plasmid RSF 1030 that includes the origin of DNA replication was determined using the DNA of a small derivative, pST19. The nucleotide sequence of the pST 19 origin region is very similar to that of the ColE1 origin except for a 25 base pair (bp) deletion about 350 bp upstream of the origin and a considerable difference in the region between 400 and 600 bp upstream of the origin. Replication of pST19 starts at one of three consecutive nucleotides (dA, dA or dC) located at a unique position in the region where the nucleotide sequence is identical to that of the ColE1 origin. There are two major sites of initiation of transcription in the region. Transcription from one of the sites yields the primer precursor that can be cleaved by RNase H to form the primer of about 530 nucleotides long. Transcription from the other site proceeds on the opposite strand and terminates close to the primer initiation site to yield species I RNA (or RNA I) about 105 nucleotides long. The presumed RNA polymerase binding sites in the promoters of these transcripts differ from those of the corresponding ColE1 transcripts. Incompatibility specified by pST19 is different from that specified by ColE1. Hypothetical peptides encoded by the origin region of these plasmids are unlikely to be involved in the determination of incompatibility. It has been shown that RNA I is an incompatibility-group specific inhibitor of primer formation. Despite a significant difference in nucleotide sequence, the primer RNA and RNA I of pST19 can be folded into structures analogous to those of the ColE1 transcripts.

Base Sequence↗
PubMed1997

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1995

Expression of human cytomegalovirus UL36 and UL37 genes is required for viral DNA replication.

It was previously reported that the region encoding human cytomegalovirus (HCMV) genes UL36 to UL38 was required for origin-dependent DNA replication. These genes encode transactivators that upregulate viral and cellular transcription. However, their requirement for viral DNA replication has not been demonstrated. We have now used an antisense phosphorothioate oligonucleotide complementary to the intron-exon boundary of the UL36 and UL37 unspliced RNA to show that these gene products are required for HCMV DNA replication. Southern analysis showed that this oligonucleotide almost completely inhibits HCMV DNA replication when used at concentrations as low as 0.08 microM. The ability of this oligonucleotide to inhibit DNA replication was not the result of an inhibition of virus adsorption. Southern blots showed no impairment of viral adsorption or internalization in the presence of either specific or nonspecific phosphorothioate oligonucleotides. In addition, Northern (RNA) blots confirm that this antisense compound specifically reduced UL36 mRNA in treated cells to undetectable levels while the steady-state levels of immediate-early transcripts IE1 and IE2 were unaffected. These results demonstrate that the UL36 and UL37 gene products provide an essential function in initiation of HCMV DNA replication.

Amino Acid Sequence↗
PubMed1996

Characterization of bacterial cell membrane attachment sites of plasmid R6K.

In vitro binding studies revealed that plasmid R6K could attach to both inner and outer membrane fractions of its host cell, Escherichia coli. Derivatives of R6K carrying one or two of its three origins of replication could not bind stably to the same membrane fractions in the presence of salt. However, the derivative, pRK35, carrying the intact three origins of replication could bind stably to membrane fractions from its host in the presence or absence of salt. These observations suggest that the three origins of DNA replication must be contiguous for stable binding of the plasmid to the cell membrane. The results of binding experiments showed that plasmid R6K bound competitively with pRK35 as well as the heterologous plasmid, pl524.

Binding, Competitive↗
PubMed1998

GATC motifs may alter the conformation of DNA depending on sequence context and N6-adenine methylation status: possible implications for DNA-protein recognition.

As part of our analysis of the role of a uniquely clustered set of dam methylation sites (the motif GATC) within the origin of DNA replication in Escherichia coli, we have studied the effect of GATCs in various methylation states on the intrinsic curvature of DNA. We have designed a set of DNA linkers and used commercially available linkers containing GATC motifs. The linkers were ligated and the electrophoretic mobility of the resulting multimers in different states of methylation was tested relative to reference fragments. We report that properly phased GATCs in certain sequence environments modulate DNA curvature and that these effects may be enhanced by N6-adenine methylation of the GATCs. These structural alterations may in turn affect DNA-protein interactions, especially those involving proteins that rely on both primary sequence and structure for recognition. We present an example, where introduction of a GATC within an integration host factor (IHF) binding site, which does not alter the consensus sequence, reduces the binding affinity of the protein for the modified site.

Adenine↗
PubMedDate not supplied

Reduction of the minimal sequence for initiation of DNA synthesis by qualitative or quantitative changes of an initiator protein.

Initiation of DNA synthesis at an origin of DNA replication involves complex protein-DNA interactions that are still poorly understood. Some of these interactions are highly specific and involve proteins (initiator proteins) thought to be essential for regulation of the initiation process because of their rate-limiting activity. We show here that both qualitative and quantitative changes in one of these proteins have profound effects on protein-DNA interactions at an origin of DNA replication, and are sufficient to reduce to less than one-third the minimal sequence required for initiation. The general implications of these findings are discussed.

Bacteriophages↗
PubMed1985

Structure of simian virus 40-adeno-associated virus recombinant genomes.

The structures of recombinant genomes formed by recombination between simian virus 40 (SV40) and adeno-associated virus 2 (AAV) DNAs after either DNA cotransfection or coinfection by virions were characterized. Two types of structures were found. Group A structures, found after cotransfection and in one of seven recombinants arising from coinfection, represented a simple deletion of SV40 sequences replaced by a slightly shorter AAV sequence. Group B structures were found in six of seven recombinants arising after virion coinfection. All contained either the left or right terminal sequences (approximately 250 to 450 bases) of the AAV genome adjacent to the SV40 origin of DNA replication. Only 350 to 650 bases (including the origin) remained of the SV40 sequence. The joined SV40-AAV sequences were present in the recombinant genome as a tandem repeat of a size that can be packaged into SV40 capsids.

Base Sequence↗
PubMed1988

The replicative origin of the E. coli chromosome binds to cell membranes only when hemimethylated.

DNA from the E. coli replicative origin binds with high affinity to outer membrane preparations. Specific binding regions are contained within a 463 bp stretch of origin DNA between positions -46 and +417 on the oriC map. This region of DNA contains an unusually high number of GATC sites, the recognition sequence for the E. coli DNA adenine methylase. We show here that oriC DNA binds to membrane only when it is hemimethylated. The E. coli chromosomal origin is hemimethylated for 8-10 min after initiation of replication, and origin DNA binds to membranes only during this time period. Based on these results, we propose a speculative model for chromosome segregation in E. coli.

Cell Membrane↗
PubMed1993

Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system.

Here a method is described to identify genes encoding proteins that recognize a specific DNA sequence. A bank of random protein segments tagged with a transcriptional activation domain is screened for proteins that can activate a reporter gene containing the sequence in its promoter. This strategy was used to identify an essential protein that interacts in vivo with the yeast origin of DNA replication. Matches between its predicted amino acid sequence and peptide sequence obtained from the 50-kilodalton subunit of the yeast origin recognition complex (ORC) established that the gene isolated here, ORC6, encodes this subunit. These observations provide evidence that ORC recognizes yeast replication origins in vivo.

Amino Acid Sequence↗
PubMed1996

Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome.

A yeast autonomously replicating sequence, ARS305, shares essential components with a chromosome III replicator, ORI305. Known components include an ARS consensus sequence (ACS) element, presumed to bind the origin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element. Here linker substitution mutagenesis of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence. The major functional element resides directly 3' of the ACS and the two remaining elements reside further downstream, all within non-conserved ARS sequences. To determine the contribution of the elements to replication origin function in the chromosome, selected linker mutations were transplaced into the ORI305 locus and two-dimensional gel electrophoresis was used to analyze replication bubble formation and fork directions. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome. Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels. Thus multiple DNA elements identified in the plasmid ARS are determinants of replication origin function in the natural context of the chromosome. Comparison with two other genetically defined chromosomal replicators reveals a conservation of functional elements known to bind ORC, but no two replicators are identical in the arrangement of elements downstream of ORC binding elements or in the extent of functional sequences adjacent to the ACS.

Base Sequence↗
PubMedDate not supplied

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein.

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.

Antigens, Viral, Tumor↗
PubMed2004

The B-subunit of DNA polymerase alpha-primase associates with the origin recognition complex for initiation of DNA replication.

The B-subunit (p70/Pol12p) of the DNA polymerase alpha-primase (Polalpha-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polalpha-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polalpha-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polalpha-primase in the initiation of both leading and lagging strands at the replication origins.

Animals↗
PubMed2000

The unusual gene organization of Leishmania major chromosome 1 may reflect novel transcription processes.

The complete chromosomal sequence for chromosome 1 from Leishmania major Friedlin predicts that this chromosome has 79 protein-coding genes. Surprisingly, the first 29 of these genes are encoded in tandem on one strand of DNA, and the remaining 50 genes are encoded in tandem on the other. No RNA polymerase promoters, centromeric sequences or origins of DNA replication have been identified in the DNA sequence. Statistical analyses of the nucleotide content reveal striking, non-random, sequence-biases that are correlated with genome organization. Analysis of coding regions suggests that novel transcription processes in Leishmania may be responsible for the nucleotide bias, which in turn affects gene organization in the chromosome. These results also suggest that the region between the two units of in-tandem genes is a candidate for an origin of DNA replication.

Algorithms↗
PubMed1987

Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template.

Analysis of the kinetics of simian virus 40 (SV40) DNA replication in vitro demonstrated the existence of a slow presynthesis reaction that occurs prior to onset of extensive chain elongation and is dependent on a subset of the cellular proteins required for the complete replication reaction. When the presynthesis reaction is carried out in the presence of topoisomerase I, it is possible to detect extensive unwinding of the template DNA. This unwinding reaction is specific for templates that contain the wild-type SV40 origin of DNA replication and requires SV40 large tumor antigen (T antigen), ATP, and a protein fraction derived from HeLa cells. The required cellular protein may be a eukaryotic single-stranded-DNA-binding protein (SSB), since unwinding of the template is also observed when Escherichia coli SSB is substituted for the HeLa protein fraction. These observations suggest that during the initial stages of SV40 DNA replication, T antigen binds specifically to the viral origin and locally unwinds the DNA. This origin-dependent unwinding reaction is presumably a prerequisite for subsequent priming and elongation steps.

Antigens, Polyomavirus Transforming↗
PubMed2000

Replication of oriJ-based plasmid DNA during the stringent and relaxed responses of Escherichia coli.

The oriJ-based plasmids contain the origin of DNA replication from the cryptic Rac prophage, present in the chromosomes of most Escherichia coli K-12 strains. The organization of the oriJ replication region resembles that of the bacteriophage lambda, although sequence similarity is small. Here we investigated the regulation of replication of the oriJ-based plasmid in E. coli relA(+) and relA(-) hosts during amino acid starvation and limitation, i.e., during the stringent and relaxed responses. We found that, contrary to plasmids derived from phage lambda, replication of the oriJ-based plasmid proceeds efficiently during both stringent and relaxed responses. On the other hand, density shift experiments and measurement of the stability of a putative replication initiator protein (the lambda O protein homologue) suggest that this replication may be carried out by the heritable replication complex, as previously demonstrated for lambda plasmids. We demonstrate that contrary to bacteriophage lambda p(R) promoter, an analogous promoter from the oriJ region is activated rather than inhibited at increased ppGpp levels. We propose that various responses of these promoters (p(R) and p(R-Rac), which are necessary for transcriptional activation of orilambda and perhaps oriJ, respectively) to ppGpp are responsible for differences in the replication regulation between orilambda- and oriJ-based plasmids during the stringent response.

Artificial Gene Fusion↗
PubMed1998

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

Binding Sites↗