Distal Sequences, but Not ori-β/OBR-1, Are Essential for Initiation of DNA Replication in the Chinese Hamster DHFR Origin

Kalejta, Robert F.; Li, X; Mesner, Larry D.; Dijkwel, Pieter A.; Lin, H.-B.; Hamlin, Joyce L.

doi:10.1016/s1097-2765(00)80294-4

Cited by 86 publications

(105 citation statements)

References 60 publications

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“…In Figure 7A and B, the 2D gel results for ori-␤ in the promoterless ⌬Pml-lox cell line are compared with DR-8, a hemizygous DHFR-deficient variant lacking the 3Ј-end of the DHFR gene (Jin et al 1995). In DR-8, there is almost a total lack of replication intermediates at 90 min, both at ori-␤ and in the body of the DHFR gene, regardless of film exposure times or amounts of material loaded onto the gels (Kalejta et al 1998;L.D. Mesner, unpubl.; also see Discussion).…”

Section: The Replication Profiles Of the Dhfr Gene In The Deletion Vamentioning

confidence: 99%

“…1D) were transfected into DG22 cells by electroporation as described (Kalejta et al 1998). Transfectants were plated in MEM containing 10% FCII supplemented with hypoxanthine and thymidine (HT; ∼3-5 × 10 6 cells per 10-cm culture dish).…”

Section: Generating Deletion Variants By Homologous Recombinationmentioning

confidence: 99%

“…The DHFR initiation zone in Chinese hamster ovary (CHO) cells was the first mammalian origin identified (Heintz and Hamlin 1982;Heintz et al 1983), and is presently the most thoroughly characterized of the several mammalian origins that have since been localized (Leu and Hamlin 1989;Vaughn et al 1990;Dijkwel and Hamlin 1995;Pelizon et al 1996;Kalejta et al 1998;Kobayashi et al 1998;Mesner et al 2003). This origin consists of a zone of >20 potential initiation sites distributed throughout the 55-kb spacer between the convergently transcribed 2BE2121 and DHFR genes (Fig.…”

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confidence: 99%

“…In previous studies, we used a homologous recombination strategy to specifically delete various subregions of the 55-kb intergenic spacer in an effort to identify any required cis-regulatory elements residing within the origin itself (Kalejta et al 1998;Mesner et al 2003). Surprisingly, the entire central 45 kb of the zone could be deleted without affecting the time at which the locus normally replicates in the S period.…”

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confidence: 99%

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The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

Saha¹,

Shan²,

Mesner³

et al. 2004

Genes Dev.

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The dihydrofolate reductase (DHFR) and 2BE2121 genes in the Chinese hamster are convergently transcribed in late G 1 and early S phase, and bracket an early-firing origin of replication that consists of a 55-kb zone of potential initiation sites. To test whether transcription through the DHFR gene is required to activate this origin in early S phase, we examined the two-dimension (2D) gel patterns of replication intermediates from several variants in which parts or all of the DHFR promoter had been deleted. In those variants in which transcription was undetectable, initiation in the intergenic spacer was markedly suppressed (but not eliminated) in early S phase. Furthermore, replication of the locus required virtually the entire S period, as opposed to the usual 3-4 h. However, restoration of transcription with either the wild-type Chinese hamster promoter or a Drosophila-based construct restored origin activity to the wild-type pattern. Surprisingly, 2D gel analysis of promoterless variants revealed that initiation occurs at a low level in early S phase not only in the intergenic region, but also in the body of the DHFR gene. The latter phenomenon has never been observed in the wild-type locus. These studies suggest that transcription through the gene normally increases the efficiency of origin firing in early S phase, but also suppresses initiation in the body of the gene, thus helping to define the boundaries of the downstream origin.

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Section: The Replication Profiles Of the Dhfr Gene In The Deletion Vamentioning

confidence: 99%

Section: Generating Deletion Variants By Homologous Recombinationmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

Saha¹,

Shan²,

Mesner³

et al. 2004

Genes Dev.

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“…Therefore, each copy must contain specific cis-acting sequences that determine where replication can begin. Moreover, replication origins exhibit initiation activity when they are translocated to other chromosomal sites (13-15), 2 and origin activity can be eliminated by deletion of specific sequences either within the initiation site or at some distance away (4,5,13,16). The distal sequences may act as enhancers to alter chromatin structure, while the proximal sequences may act as assembly sites for prereplication complexes.…”

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confidence: 99%

DNA Methylation at Mammalian Replication Origins

Rein

Kobayashi²,

Malott³

et al. 1999

Journal of Biological Chemistry

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In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the ϳ2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin ␤ (ori-␤), downstream of the hamster DHFR gene. Remethylation at ori-␤ did not begin until ϳ500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-␤ activity. Cells that were >50% reduced in methylation at ori-␤ no longer selectively activated ori-␤. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.In early embryos undergoing rapid cell cleavage (e.g. frogs, flies, sea urchin, fish), initiation of DNA replication appears neither to require specific DNA sequences nor to occur at specific DNA sites. However, as development progresses past the blastula stage, initiation of DNA replication begins to occur at specific sites (1, 2). Thus, it is not surprising that initiation sites for DNA replication in cultured mammalian cells occur at specific genomic loci (3). For example, a 200-kb 1 region at the human ␤-globin gene (4, 5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).Nevertheless, the precise size and composition of these initiation loci, as well as the parameters that define them remain the subject of intense investigation.The fact that specific sites for initiation of DNA replication can be developmentally acquired makes it clear that initiation sites in the metazoa are determined at least in part by epigenetic parameters. These parameters include nuclear structure, chromatin structure, and even...

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DNAReplication in Humans

DePamphilis¹

2002

Wiley Encyclopedia of Molecular Medicine

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Distal Sequences, but Not ori-β/OBR-1, Are Essential for Initiation of DNA Replication in the Chinese Hamster DHFR Origin

Cited by 86 publications

References 60 publications

The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

DNA Methylation at Mammalian Replication Origins

DNAReplication in Humans

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