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Results for “DNA replication origin”

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PubMed2003

Same origins of DNA replication function on the active and inactive human X chromosomes.

We previously characterized a functional origin of DNA replication at the transcriptional promoter of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Cohen et al. [2002] J. Cell. Biochem. 85:346-356). This origin was mapped using a quantitative PCR assay to evaluate the relative abundance of HPRT markers in short nascent DNA strands isolated from asynchronous cultures of male fibroblasts. The HPRT gene on the X chromosome is transcriptionally active in male human fibroblasts. It is known that on the heterochromatic X chromosome in female cells the HPRT gene is transcriptionally silenced and its replication timing changes from early to late in S phase. This change in replication timing could indicate that replication of the HPRT gene is under the control of different origins of DNA replication in the active (euchromatic, early replicating) and the inactive (heterochromatic, late replicating) X chromosomes. In the present study, we identified the location of the origin of replication of a second X chromosome gene, glucose-6-phosphate dehydrogenase (G6PD), which we mapped to its transcriptional promoter, in normal male human fibroblasts. Then, we determined the activity of the previously identified HPRT and the G6PD human origins in hybrid hamster cells carrying either the active or the inactive human X chromosome. The results of these studies clearly demonstrated that the human HPRT and G6PD origins of replication were utilized to the same extent in the active and the inactive X chromosomes. Therefore, transcription activity at the HPRT and G6PD genes is not necessary for initiation of DNA replication at the origins mapped to these chromosomal loci.

Animals↗

PubMed2002

The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication.

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat-containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.

Animals↗

PubMed2001

Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants.

The antitumor drug adozelesin is a potent cytotoxic DNA-damaging agent. Here we determined how adozelesin affects chromosomal DNA replication at a molecular level in a yeast model system and examined the influence of checkpoint kinase genes, the human homologues of which are mutated in cancer. Analysis of replication intermediates using two-dimensional gel electrophoresis showed that adozelesin inhibited the activity of a replication origin and stalled replication fork progression through chromosomal DNA at the origin. RAD53 and MEC1 protein kinase genes, homologues of human CHK2 and ATM, respectively, regulate an intra-S-phase DNA damage checkpoint and, when mutated, permit unchecked replication of damaged DNA in S-phase. Mutations in these genes did not abrogate adozelesin-induced inhibition of origin activity and fork progression at the replication origin. However, novel replication intermediates indicative of DNA breaks were detected only in the rad53 mutant, suggesting a role for the wild-type gene in maintaining chromosome integrity in the presence of the drug. In contrast to the inhibition of the active replication origin by adozelesin, normally silent origins present in the same chromosome were activated by adozelesin in rad53 and mec1 mutant cells. Thus, an antitumor drug that damages DNA can induce an abnormal replication pattern in a chromosome by activating silent origins, depending upon defects in yeast checkpoint kinase genes, the homologues of which are mutated in cancer. Implications of an abnormal replication pattern for the epigenetic regulation of gene expression are discussed.

Antineoplastic Agents, Alkylating↗

PubMed1999

The "small" polydisperse cytoplasmic extrachromosomal DNA of chicken leukaemic myeloblasts and the avian myeloblastosis virus core-bound DNA seem to descend from origin regions of chromosomal DNA replication.

Nucleotide sequences are presented for 12, 7 and 12 cloned extrachromosomal DNAs by nature harbored in nucleoprotein (NP) complexes forming chicken leukaemic myeloblast (CHLM) post microsomal sediment (POMS) components A, B and C, respectively, and for 11 cloned avian myeloblastosis virus (AMV) DNAs. Analysis of the abundance of sequence motifs significant for eukaryotic chromosomal DNA replication origin (ori) regions (and their initiation zones) has shown that these DNAs are reminiscent of cell DNA fragments enriched in ori sequences (Rao et al., 1990) and/or sequence features of several eukaryotic chromosomal oris containing clusters of modular sequence elements (Dobbs et al., 1994). Accordingly, these DNAs, with an (A + T) content prevalently higher than that of the total cell DNA, revealed the presence of asymmetrically distributed (A + T)-rich stretches, scaffold attachment region (SAR) T consensuses, polypyrimidine nucleotide (poly(Py)) tracts and minimal Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, in abundance comparable with that of these sequences of DNA fragments enriched in oris. All these DNAs were found to be enriched also in sequence elements held as primase (Pr) attachment sites. Moreover, DNAs of POMS component B and those of AMV DNA were found to be enriched in the asymmetric pyrimidine (Py) heptanucleotide motif of Waltz et al. (1996) occurring in the initiation zones of ori region. Consequently, these extrachromosomal DNAs, portion of which represents a precursor of AMV DNA, seem to descent from initiation zones of various ori regions of an early replicating chromosomal myeloblast DNA. In addition, a possible explanation of the inclination of these DNAs to form multimers is presented.

Animals↗

PubMed1995

Cellular proteins bind to the downstream component of the lytic origin of DNA replication of Epstein-Barr virus.

The lytic origin of DNA replication of Epstein-Barr virus, oriLyt, is a complex eukaryotic origin which is activated during the lytic phase of the viral life cycle. It consists of at least two independent cis-acting components, one of which plays a dual role in transcription and DNA replication. The binding of the viral factor BZLF1, a member of the AP1 family of transcription factors, to this upstream component is crucial for oriLyt function (A. Schepers, D. Pich, and W. Hammerschmidt, EMBO J. 12:3921-3929, 1993). The second cis-acting element, the downstream component of oriLyt, is equally indispensable; however, its function is unknown. In this study, the downstream component was found to be the binding target of several cellular proteins. One could be identified as Sp1 or as a related protein which binds twice to the downstream component of oriLyt. Mutational analysis indicated that Sp1 alone is not directly involved in mediating DNA replication; however, other factors which share the same binding sequence or bind closely to one of the Sp1 binding sites are likely candidates to contribute to a replication protein complex at the downstream component of oriLyt. The sequence requirements for the downstream component are remarkably stringent, indicating that at least one of the putative factors is a sequence-specific DNA-binding protein which is required for the activation of oriLyt.

Base Sequence↗

PubMed1982

Identification of the origins of T4 DNA replication.

Two physical origins of T4 DNA replication were determined by hybridization of viral DNA prepared 2.5 min after infection to a display of total T4 DNA. This is the earliest time after T4 infection of Escherichia coli at 37 degrees C that labeled and hybridizable DNA can be detected. The two origins, separated by about 25 kilobases, were identified and localized in the early region of the T4 map. One of them is located in a 5.6-kilobase EcoRI fragment containing genes 62-46. The other is located between genes rI and e in a 1.9-kilobase EcoRI fragment. Both of these T4 fragments have been cloned and their interactions with the host cell are discussed.

Base Sequence↗

PubMed1998

The Cdc7 protein kinase is required for origin firing during S phase.

The Cdc7p protein kinase plays an essential, but undefined, role promoting S phase in the budding yeast, Saccharomyces cerevisiae. Previous experiments have shown that the essential function of Cdc7 is executed near the G1-S boundary; after Start but before the elongation phase of DNA replication. Origins of DNA replication fire throughout S phase in budding yeast. Therefore, the G1-S transition is a cell-cycle event that precedes, and is distinct from, the activation of individual origins. Consequently, we have asked whether Cdc7 is only required for S-phase entry or if it plays a role during S phase in origin firing. In this article, we show that partial loss of Cdc7 function results in slow progression through S phase rather than slow entry into S phase and that Cdc7 is still required for the timely completion of S phase after a block to elongation with hydroxyurea. This is because Cdc7 is still required for the activation of late-firing origins after the hydroxyurea block. These experiments show that, rather than acting as a global regulator of the G1-S transition, Cdc7 appears to play a more direct role in the firing of replication origins during S phase.

Cell Cycle Proteins↗

PubMed1998

In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication.

DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro.

Animals↗

PubMed2004

Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model.

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.

Animals↗

PubMed1994

Early dissociation of nuclear factor I from the origin during initiation of adenovirus DNA replication studied by origin immobilization.

The DNA-binding domain of Nuclear Factor I (NFIBD) enhances initiation of adenovirus DNA replication up to 50-fold by binding to the auxiliary region of the origin and positioning the viral DNA polymerase. To study if and when NFIBD dissociates from the template, we immobilized origin DNA to glutathione-agarose beads by means of a GST-NFIBD fusion protein. This immobilized template is active in replication. By analyzing the release of prelabeled templates from the beads under different conditions, we show that NFIBD dissociates already early during initiation. During preinitiation NFIBD remains bound, but as soon as dCTP, dATP or dTTP are added, efficient dissociation occurs. A much lower dissociation level was induced by addition of dGTP. Since dCTP, dATP and dTTP are required for formation of a pTP-CAT initiation intermediate, we explain our results by conformational changes occurring in the polymerase during initiation leading to disruption of both the interaction between the polymerase and NFI as well as the interaction between NFI and the DNA.

Adenoviridae↗

PubMed2002

Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45.

Current models suggest that the replication initiation factor Mcm10 is required for association of Mcm2-7 with origins of replication to generate the prereplicative complex (pre-RC). Here we report that Xenopus Mcm10 (XMcm10) is not required for origin binding of XMcm2-7. Instead, the chromatin binding of XMcm10 at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7. In the absence of XMcm10, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked. Therefore, XMcm10 performs its function after pre-RC assembly and before origin unwinding. As one of the earliest known pre-RC activation steps, chromatin binding of XMcm10 is an attractive target for regulation by cell cycle checkpoints.

Animals↗

PubMed1991

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2.

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.

Bacterial Proteins↗

PubMed2000

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes.

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.

Base Sequence↗

PubMed1992

ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.

A multiprotein complex that specifically recognizes cellular origins of DNA replication has been identified and purified from the yeast Saccharomyces cerevisiae. We observe a strong correlation between origin function and origin recognition by this activity. Interestingly, specific DNA binding by the origin recognition complex is dependent upon the addition of ATP. We propose that the origin recognition complex acts as the initiator protein for S. cerevisiae origins of DNA replication.

Adenosine Triphosphate↗

PubMed1999

Comparative characterization of rep proteins from the helper-dependent adeno-associated virus type 2 and the autonomous goose parvovirus.

Adeno-associated viruses (AAVs) are nonautonomous human parvoviruses in that they are dependent on helper functions supplied by other viruses or on genotoxic stimuli for conditions permissive for replication. In the absence of helper, AAV type 2 enters latency by integration into a specific site on human chromosome 19. This feature of AAV, in combination with a lack of pathogenicity, makes AAV an attractive candidate vector for human gene therapy. Goose parvovirus (GPV) is both autonomous and pathogenic yet is highly homologous to AAV. To address the molecular bases for the different viral lifestyles, we compare the AAV and GPV nonstructural proteins, Rep78 and Rep1, respectively. We find that Rep78 and Rep1 possess several biochemical activities in common, including (i) high-affinity DNA binding for sequences that constitute the minimal DNA replication origin; (ii) nucleoside triphosphate-dependent DNA helicase activity; and (iii) origin-specific replication of double-stranded linear DNA. These experiments also establish a specific 38-bp DNA sequence as the minimal GPV DNA replication origin. It is noteworthy that although the proposed Rep binding sites of GPV and AAV are highly similar, Rep1 and Rep78 show a high degree of specificity for their respective origins, in both binding and replication assays. One significant difference was observed; with the minimal replication origin in adenovirus-uninfected extracts, Rep78-mediated replication exhibited low processivity, as previously reported. In contrast, Rep1 efficiently replicated full-length template. Overall, our studies indicate that GPV Rep1 and AAV Rep78 support a comparable mode of replication. Thus, a comparison of the two proteins provides a model system with which to determine the contribution of Rep in the regulation of dependence and autonomy at the level of DNA replication.

Animals↗

PubMed1995

Stimulation of DNA synthesis by mouse DNA helicase B in a DNA replication system containing eukaryotic replication origins.

A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far. We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin. In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin. We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen. DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase. DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A. A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B. Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system.

Adenosine Triphosphatases↗

PubMed1999

Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control.

The mechanisms controlling duplication of the metazoan genome are only beginning to be understood. It is still unclear what organization of DNA sequences constitutes a chromosomal origin of DNA replication, and the regulation of origin activity during the cell cycle has not been fully revealed. We review recent results that indicate that chorion gene amplification in follicle cells of the Drosophila ovary is a model for investigating metazoan replication. Evaluation of cis sequence organization and function suggests that chorion loci share attributes with other replicons and provides insights into metazoan origin structure. Moreover, recent results indicate that chorion origins respond to S-phase control, but escape mechanisms that inhibit other origins from firing more than once in a cell cycle. Several identified genes that mediate amplification are critical for the cell cycle control of replication initiation. It is likely that further genetic screens for mutations that disrupt amplification will identify the cadre of proteins associated with origins and the regulatory pathways that control their activity. Furthermore, the recent development of methods to detect amplification in situ has uncovered new aspects of its developmental control. Examining this control will reveal links between developmental pathways and the cell cycle machinery. Visualization of amplifying chorion genes with high resolution also represents an opportunity to evaluate the influence of nuclear and chromosome structure on origin activity. The study of chorion amplification in Drosophila, therefore, provides great potential for the genetic and molecular dissection of metazoan replication.

Animals↗

PubMed1996

Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication.

During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells. It is not known, however, what the early events in viral DNA replication that yield these concatemers are. In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed. As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA). A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus. This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction. The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids. Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication.

Cell Line↗