DNA replication from initiation zones of mammalian cells in a model system.

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An important relationship between transcription and initiation of DNA replication in both eukaryotes and prokaryotes has been suggested. In an attempt to understand the molecular mechanism of this interaction, we examined whether transcription can induce DNA replication in vitro by constructing a system in which both replication and transcription were combined. Relaxed circular DNA possessing a replication initiation zone located upstream of the human c-myc gene and a T7 promoter near the P1 promoter of the gene was replicated in the presence of T7 RNA polymerase. In our model system, replication was carried out with the proteins required for simian virus 40 DNA replication. DNA synthesis, which was dependent on both T7 RNA polymerase and the replication proteins, was detected mainly in the promoter and upstream regions of the c-myc gene. Blocking RNA synthesis at the initial stage of the reaction severely reduced DNA synthesis, suggesting that RNA chain elongation is required to induce DNA synthesis. The results indicated that transcription can induce DNA replication in the upstream region of the transcribed gene, most likely by introducing negative supercoiling into the region, which results in unwinding of the DNA duplex.DNA replication in prokaryotes and eukaryotic viral systems is significantly affected by transcription and/or transcription factors (11, 37). In Escherichia coli, transcription of the gid gene, located on the left side of the replication origin, stimulates oriC plasmid DNA replication (1). In polyomavirus-infected cells, the binding of the transcription factor AP1 to the flanking region of the origin markedly stimulates viral DNA replication in vivo (19, 41). Nuclear factor I (NFI) (42) and NFIII (45), both of which are required for adenovirus DNA replication in vitro, are identical to the cellular transcription factors CTF (47) and OCT1 (44), respectively. The binding site for the transcription factor ABF1 (12), which constitutes the B3 domain of ARS1, is required for DNA replication in Saccharomyces cerevisiae (36). In higher eukaryotes, nearly all the actively transcribed genes are replicated early in S phase whereas inactive genes are replicated late in S phase (17, 23). Colocalization of transcription and DNA replication in nuclei has been shown in permeabilized HeLa cells (22). However, the molecular mechanisms underlying the relationship between transcription and replication remain unclear.Both specific sequences in the origin and initiator proteins that interact with these sequences are essential for the initiation of DNA replication in prokaryotes (9) and in eukaryotic viruses (8). In S. cerevisiae, both essential origin sequences (10) and a protein complex (3) that binds to the origin sequences have been identified. While short sequences essential for DNA replication have not been found in higher eukaryotes, an increasing number of regions in the chromosome in which DNA replication initiates have been mapped and are called initiation zones (5,20,24). Several of these have been ident...

Section: Discussionsupporting

confidence: 66%

supporting

confidence: 82%

mentioning

confidence: 71%

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Induction of DNA Replication by Transcription in the Region Upstream of the Human c-myc Gene in a Model Replication System

Ohba

Matsumoto

Ishimi

1996

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“…However, neither the mammalian initiator protein(s) nor the helicase(s) specific for DNA replication has been definitively identified (23,25). Moreover, the exact origins of mammalian DNA replication are also not defined and appear to reside in initiation zones rather than consist of precise DNA consensus sequences (27,29). To circumvent these obstacles and still obtain valuable information regarding the early stages of mammalian cell DNA replication, we are exploiting the simian virus 40 (SV40) model of DNA replication in vitro, which depends on a well-defined origin of replication and a single initiator protein and DNA helicase, the virally encoded T antigen (10,35,59).…”

mentioning

confidence: 99%

Human Topoisomerase I Promotes Initiation of Simian Virus 40 DNA Replication In Vitro

Trowbridge

Roy

Simmons

1999

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.A major area of interest under investigation is the molecular mechanism involved in the early stages of mammalian cell DNA replication. However, neither the mammalian initiator protein(s) nor the helicase(s) specific for DNA replication has been definitively identified (23,25). Moreover, the exact origins of mammalian DNA replication are also not defined and appear to reside in initiation zones rather than consist of precise DNA consensus sequences (27,29). To circumvent these obstacles and still obtain valuable information regarding the early stages of mammalian cell DNA replication, we are exploiting the simian virus 40 (SV40) model of DNA replication in vitro, which depends on a well-defined origin of replication and a single initiator protein and DNA helicase, the virally encoded T antigen (10,35,59). All essential replication machinery components except T antigen are provided by permissive cellular extracts (34,35,59,68). Further studies have led to the establishment of reconstituted systems from purified proteins that support SV40 DNA replication (26,64,65,69). Use of these model systems has provided a wealth of information regarding the functions of proteins involved in eukaryotic DNA replication (recently reviewed in references 5 and 20). However, there are still significant gaps in our understanding of the exact mechanisms involved in eukaryotic DNA replication, in particular with respect to (i) the composition and geometry of the initiation and elongation complexes, (ii) the interactions among replication factors, and (iii) how replication is regulated at various stages.T antigen provides the helicase activity by forming a double hexamer over the SV40 origin of replication in the presence of ATP (3,11,38). In a...

“…Naked DNA contains many potential sites where replication can begin. These sites most likely correspond to easily unwound DNA sequences that are components of replication origins in simple (75) as well as metazoan (33) genomes and that can promote site-specific replication in plasmid DNA (51). When naked DNA is introduced into Xenopus eggs or egg extract, chromatin is assembled in the absence of histone H1, which is required for compaction of DNA into 30-nm fibers (94), and in the presence of only one nuclear lamin protein, a component of nuclear structure required for DNA replication (53,78), instead of the three found in differentiated cells (5,86).…”

mentioning

confidence: 99%

Site-Specific Initiation of DNA Replication in Xenopus Egg Extract Requires Nuclear Structure

Gilbert

Miyazawa

DePamphilis

1995

117

184

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G 1 -phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G 1 -phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-␤). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-␤ was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-␤. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.Origins of DNA replication in animal viruses and single-cell eukaryotic organisms consist of specific cis-acting sequences that function independently (autonomously replicating sequences) when transferred to the appropriate cells or cell extract (25,26,58). However, attempts to identify similar sequences in multicellular eukaryotes have resulted in a paradox: while DNA synthesis is initiated at specific, genetically determined sites in cellular chromosomes, identification of cis-acting DNA sequences that control replication has proven elusive.Mapping initiation sites for DNA replication at 17 different locations in the chromosomes of mammals and flies has revealed that DNA synthesis initiates not randomly throughout cellular chromosomes but at specific DNA sites. These sites consist of a primary initiation locus referred to as the origin of bidirectional replication (OBR) surrounded by many secondary initiation loci distribute...