Identification of an Origin of Bidirectional DNA Replication in the Ubiquitously Expressed Mammalian <i>CAD</i> Gene

Kelly, Ruth E.; Rose, M; Draper, Bruce W.; Wahl, Geoffrey M.

doi:10.1128/mcb.15.8.4136

Cited by 39 publications

(32 citation statements)

References 126 publications

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“…Cells lacking wt-p 53 form colonies in PALA at high frequency, presumably through a mechanism in which the CAD gene is amplified (Livingstone et al, 1992;Yin et al, 1992;Schaefer et al, 1993;Kelly et al, 1995). The CAD gene encodes a single polypeptide chain containing carbamyl phosphate synthetase, dihydroorotase and aspartate transcarbamylase (which PALA inhibits).…”

Section: Enhancement Of Cad Gene Amplification In Cells Expressing Onmentioning

confidence: 99%

Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras

et al. 1998

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Section: Enhancement Of Cad Gene Amplification In Cells Expressing Onmentioning

confidence: 99%

Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras

et al. 1998

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“…Most OBRs are contained within as little as 0.5 kb to as much as 3 kb (references 4, 6, 26, 39, 64, 87, 88, 91, and 95 and references therein), although some OBRs lie within larger regions of 5 to 11 kb (36,46,54,83). The fact that these OBRs have been identified by independent investigators using a variety of different methods gives confidence that site-specific initiation is not an artifact of the experimental conditions used to map them.…”

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confidence: 99%

“…This conclusion is based on the fraction of replication forks traveling in the same direction as determined by two-dimensional (2D) neutral/alkaline gels (64, 83), the ratio of Okazaki fragments that hybridize to the two strands of a unique DNA probe (6,15,20,54,88), and the ratio of long leading nascent DNA strands from forward arms of replication forks that hybridize to the two strands of a unique DNA probe (16,46,54,57). In addition, quantitative analysis of specific DNA sequences within long nascent DNA strands reveals that most of them originate bidirectionally from a small chromosomal locus (39) and that this locus can reside within an initiation zone (97).Most OBRs are contained within as little as 0.5 kb to as much as 3 kb (references 4, 6, 26, 39, 64, 87, 88, 91, and 95 and references therein), although some OBRs lie within larger regions of 5 to 11 kb (36,46,54,83). The fact that these OBRs have been identified by independent investigators using a variety of different methods gives confidence that site-specific initiation is not an artifact of the experimental conditions used to map them.…”

mentioning

confidence: 99%

“…From 80 to 95% of DNA synthesis occurs bidirectionally from the OBR. This conclusion is based on the fraction of replication forks traveling in the same direction as determined by two-dimensional (2D) neutral/alkaline gels (64, 83), the ratio of Okazaki fragments that hybridize to the two strands of a unique DNA probe (6,15,20,54,88), and the ratio of long leading nascent DNA strands from forward arms of replication forks that hybridize to the two strands of a unique DNA probe (16,46,54,57). In addition, quantitative analysis of specific DNA sequences within long nascent DNA strands reveals that most of them originate bidirectionally from a small chromosomal locus (39) and that this locus can reside within an initiation zone (97).…”

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confidence: 99%

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

Gilbert

Miyazawa

DePamphilis

1995

Molecular and Cellular Biology

117

184

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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...

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“…This origin lies just upstream of the c-myc promoter and is located within a CpG island (17-20, 46 -48), typical of a class of replication origins that map to promoter regions of actively transcribed genes (18) that replicate at the beginning of S phase (49). CpG islands are regions of ϳ1 kb that contain more than 50% G and C residues and a ratio of observed/expected CpGs of greater than 0.6 (11). Although CpG islands are considered devoid of 5-methylcytosine (50), attempts to evaluate the methylated state of these origins have so far relied either on methylation-sensitive restriction endonucleases that sample only a small subset of cytosines (50) or on sequence analysis of only a small fraction of the total number of CpGs within the island (28).…”

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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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Identification of an Origin of Bidirectional DNA Replication in the Ubiquitously Expressed Mammalian CAD Gene

Cited by 39 publications

References 126 publications

Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras

Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras

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

DNA Methylation at Mammalian Replication Origins

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