High-Resolution Mapping of the Origin of DNA Replication in the Hamster Dihydrofolate Reductase Gene Domain by Competitive PCR

Pelizon, Cristina; Diviacco, Silvia; Falaschi, Arturo; Giacca, Mauro

doi:10.1128/mcb.16.10.5358

Cited by 78 publications

(93 citation statements)

References 44 publications

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“…The reliability of this approach for ORI identification is supported by the fact that several mammalian ORIs that were initially identified by a variety of non-PCR approaches have been validated later by Q-PCR. Examples include the preferential initiation sites within the disperse initiation region downstream from the hamster DHFR gene identified by two-dimensional electrophoresis (Pelizon et al 1996;Kobayashi et al 1998), the human GM-CSF Ori1 and Ori2 identified by subtractive hybridization for origin capture (Todorovic et al 2005), and several human ORIs recently identified by high-throughput array hybridization (Lucas et al 2007). Conversely, ORIs initially identified by Q-PCR have been shown to bind ORC and MCM proteins as in the case of the human Lamin B2 (Abdurashidova et al 2003), c-Myc , and FMR1 (Gray et al 2007).…”

Section: Discussionmentioning

confidence: 99%

Overreplication of short DNA regions during S phase in human cells

Gómez¹,

Antequera²

2008

Genes Dev.

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DNA replication origins (ORI) are regulatory regions from which the genome is replicated once every cell cycle. A widely used method for their identification in mammalian chromosomes relies on quantitative PCR of DNA nascent strands across candidate regions. We developed a new high-resolution PCR strategy to localize ORIs directly on total unfractionated human DNA. The increase in sensitivity provided by this approach has revealed that a short region of ∼200-base-pair overlapping well-characterized replication origins undergoes several rounds of replication, coinciding with their specific time of activation during S phase. This process generates a population of discrete dsDNA fragments detectable as free molecules in preparations of total DNA in normally proliferating cells. Overreplicated regions have precise boundaries at the edge of the nucleosome-free gap that encompasses the transcription initiation sites of CpG island promoters. By itself, active transcription does not induce overreplication but does stimulate it at ORIs associated with promoters. The coincidence in time and space between the overproduction of short DNA fragments and ORI activity predicts the precise localization of thousands of ORIs in the human genome and uncovers a previously unnoticed step in the initiation of DNA replication.[Keywords: Replication origins; CpG islands; promoters; transcription; cell cycle; chromatin structure] Supplemental material is available at http://www.genesdev.org.

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Section: Discussionmentioning

confidence: 99%

Overreplication of short DNA regions during S phase in human cells

Gómez¹,

Antequera²

2008

Genes Dev.

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“…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.…”

mentioning

confidence: 99%

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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“…Competitors were constructed essentially as previously described (71). Briefly, PCR amplification was performed with pMCD template using the standard primer sets 8,2, 6, and 3 and their corresponding competitor (COM) primer sets, which contained a 20 bp insertion (Table 1).…”

Section: Construction Of Pcr Competitive Templatesmentioning

confidence: 99%

Genetic Analysis of a Mammalian Chromosomal Origin of Replication

Altman¹

2002

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High-Resolution Mapping of the Origin of DNA Replication in the Hamster Dihydrofolate Reductase Gene Domain by Competitive PCR

Cited by 78 publications

References 44 publications

Overreplication of short DNA regions during S phase in human cells

Overreplication of short DNA regions during S phase in human cells

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

Genetic Analysis of a Mammalian Chromosomal Origin of Replication

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