On the nature of origins of DNA replication in eukaryotes

Benbow, Robert M.; Zhao, Jiapeng; Larson, Drena D.

doi:10.1002/bies.950141004

Cited by 65 publications

(46 citation statements)

References 58 publications

(30 reference statements)

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“…This region was shown to contain a strong promoter for the downstream transcription unit; a key feature ofthis promoter is the presence of a basic helix-loop-helix protein binding motif that was shown to bind in vitro to the USF/MLTF protein (23); interestingly, such motif also fits the binding consensus for the Myc/Max complex (24), whose function in regulating cell proliferation is suggested by several studies (25) [incidentally, the c-myc gene domain is amplified in HL-60 cells (26)]. An evolutionary conserved (A+T)-rich region (22) can be envisaged in the proximity of the origin, as is the case for well-defined replication origins of different organisms (27)(28)(29).…”

Section: ~-F )W Lmentioning

confidence: 81%

Fine mapping of a replication origin of human DNA.

Giacca

Zentilin

Norio

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

179

202

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A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of ammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA framents distributed along a given genomic region. Terminal differentiation of HL-60 was achieved with retinoic acid and dimethylformamide, as described (17).Transfection. Plasmid pAWTSV (=9 kb), a kind gift of Cesare Vesco (Institute of Cell Biology, Rome), carries the whole simian virus 40 (SV40) genome inserted in the BamHI site of pAT153 (18). Six 10-cm tissue culture plates, containing about 106 COS-1 cells each, were transfected with 10 pg of pAWTSV by the calcium phosphate precipitation technique. After 10 hr of incubation in calcium phosphate solution, cells were extensively washed and fresh medium was added, containing 10 nCi of [14C]thymidine per ml. After 18 hr of incubation, BrdUrd (100 uM final concentration) and[3H]deoxycytidine (1 jAM final concentration) were added.After 1 min of incubation, cells were killed by addition of sodium azide and DNA was extracted as described below.Extrction and Purification of Newly Syntez DNA.Total DNA was extracted, denatured, and size-fractionated by sedimentation through neutral sucrose gradients as described (15).In the experiment involving transfection of plasmid pAW-TSV, DNA (700 /4 final volume) was fractionated on four 5-20%6 (wt/vol) linear sucrose gradients (5 ml each) for 210 min at 200C in a Beckman SW55Ti rotor at 55 krpm; 24 fractions of 200 j4 were collected.In the experiment with synchronized HL-60 cells, DNA (2 ml final volume) was fractionated on eight 5-30% sucrose Abbreviations: DHFR, dihydrofolate reductase; SV40, simian virus 40. tTo whom reprint requests should be addressed. 7119The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: ~-F )W Lmentioning

confidence: 81%

Fine mapping of a replication origin of human DNA.

Giacca

Zentilin

Norio

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

179

202

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“…SC). These features may be important for the establishment of a preinitiation complex, since both DNA polymerase o-primase and HSSB, which play essential roles in the initiation of DNA replication in this system, bind preferentially to the single-stranded DNA containing pyrimidine-rich sequences (4,24 (Fig. 5A), suggesting the interaction of HSSB with the region.…”

Section: Discussionmentioning

confidence: 99%

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

1994

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We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y. Ishimi and K. Matsumoto, Proc. Natl. Acad. Sci. USA 90:5399-5403, 1993). In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system. When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase ot-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase. Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication. The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable. In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication. These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication. The mechanism of initiation is discussed.Both specific sequences in the origin that are within a few hundred base pairs in length and an initiator protein that interacts with the sequences are essential for initiation of DNA replication in prokaryotes and eukaryotic viruses (6, 11). Binding of the initiator protein to the sequences results in the unwinding of an AT-rich region in the origin, which is recognized by replication proteins, including DNA helicase. Similarly, an essential origin sequence has been found in Saccharomyces cerevisiae (10), and a protein complex that binds to the sequence has been identified (3), but the sequences essential for DNA replication have not been found in higher eukaryotes, including Schizosaccharomyces pombe. An increasing number of the regions in the chromosome where the initiation of DNA replication occurs in higher eukaryotes have been mapped, and they are called initiation zones (reviewed in reference 4). Vassilev and Johnson (40) have identified an initiation zone of 2 kb in size upstream of the human c-myc gene by origin mapping, using PCR amplification of nascent DNA. Consistent with this, it has been reported that the upstream region of the c-myc gene has activity by which the plasmid DNA containing the region can be replicated as an extrachromosomal DNA element (19,31 fragments (41). Furthermore, Burhans et al. (7) have identified a specific initi...

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“…Detailed analyses of prokaryotic and eukaryotic viral origins of replication have revealed that a prerequisite for initiation of replication is unwinding of double-stranded DNA (reviewed in references 10 and 11). Whereas the unwinding of DNA in prokaryotic and viral replication origins is dependent on sequence-specific binding of initiator proteins to the origin sequences and the unwound regions are restricted to the adjacent regions, there may be a mechanism in higher eukaryotes that can unwind DNA in a much extended region as proposed by Benbow and coworkers (7,28).…”

Section: Materiails and Methodsmentioning

confidence: 99%

DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites.

Shinomiya¹,

Ina²

1993

Mol. Cell. Biol.

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We showed previously that DNA replication initiates at multiple sites in the 5-kb histone gene repeating unit in early embryos of Drosophila melanogaster. The present report shows evidence that replication in the same chromosomal region initiates at multiple sites in tissue culture cells as well. First, we analyzed replication intermediates by the two-dimensional gel electrophoretic replicon mapping method and detected bubble-form replication intermediates for all fragments restricted at different sites in the repeating unit. Second, we analyzed bromodeoxyuridine-labeled nascent strands amplified by the polymerase chain reaction method and detected little differences in the size distribution of nascent strands specific to six short segments located at different sites in the repeating unit. These results strongly suggest that DNA replication initiates at multiple sites located within the repeating unit. We also found several replication pause sites located at 5' upstream regions of some histone genes. (41) showed that DNA replication initiates at multiple locations in a plasmid containing a 20-kb human chromosomal sequence in human cells. Existence of multiple origins of replication was also suggested for the amplified chorion genes in Drosophila cells by the 2D gel electrophoretic method (20,32).In contrast to these results, evidence for specific replication origins in higher eukaryotes has also been reported. The most striking contradiction is a discrepancy between the data on replication origins in the DHFR locus of CHO cells obtained by different experimental approaches. Although the 2D gel analysis of replication intermediates suggested the presence of multiple initiation sites distributed in a broad initiation zone as mentioned above, other workers reported that replication in this chromosomal region initiates at a specific site, as judged from nascent strand analyses (15,16,55

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On the nature of origins of DNA replication in eukaryotes

Cited by 65 publications

References 58 publications

Fine mapping of a replication origin of human DNA.

Fine mapping of a replication origin of human DNA.

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

DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites.

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