Mapping of Replication Initiation Sites in Human Ribosomal DNA by Nascent-Strand Abundance Analysis

Yoon, Yeup; Sanchez, J. Aquiles; Brun, Catherine; Huberman, Joel A.

doi:10.1128/mcb.15.5.2482

Cited by 64 publications

(48 citation statements)

References 48 publications

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“…S7). These findings provide a potential explanation for the contradictory results generated by earlier rDNA replication origin mapping endeavors (Little et al, 1993;Yoon et al, 1995;Gencheva et al, 1996;Lebofsky and Bensimon, 2005). It is possible that, owing to differences in experimental systems and design, and/or the capabilities of the methodologies involved, individual labs preferentially detected one of the two, or a combination of both, initiation patterns specific for the two classes of ribosomal genes.…”

Section: Replicated Rdna Relocates To the Nucleolar Interior And Reasmentioning

confidence: 72%

“…S7) (Little et al, 1993;Yoon et al, 1995;Gencheva et al, 1996;Lebofsky and Bensimon, 2005). The majority of studies (Yoon et al, 1995;Gencheva et al, 1996;Lebofsky and Bensimon, 2005) have made no attempt to look for potential differences between early-and late-replicating ribosomal gene units, whereas those that have addressed this issue (Little et al, 1993) have found no significant differences. To gain further insight into the distinct behavior of expressed and silent rRNA genes, I examined their replication initiation patterns by taking advantage of their different replication timing.…”

Section: Replicated Rdna Relocates To the Nucleolar Interior And Reasmentioning

confidence: 99%

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DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

Dimitrova

2011

Journal of Cell Science

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SummaryTypically, only a fraction of the ≥600 ribosomal RNA (rRNA) gene copies in human cells are transcriptionally active. Expressed rRNA genes coalesce in specialized nuclear compartments -the nucleoli -and are believed to replicate during the first half of S phase. Paradoxically, attempts to visualize replicating rDNA during early S phase have failed. Here, I show that, in human (HeLa) cells, earlyreplicating rDNA is detectable at the nucleolar periphery and, more rarely, even outside nucleoli. Early-replicated rDNA relocates to the nucleolar interior and reassociates with the transcription factor UBF, implying that it predominantly represents expressed rDNA units. Contrary to the established model for active gene loci, replication initiates randomly throughout the early-replicating rDNA. By contrast, mostly silent rDNA copies replicate inside the nucleoli during mid and late S phase. At this stage, replication origins are fired preferentially within the non-transcribed intergenic spacers (NTSs), and ongoing rDNA transcription is required to maintain this specific initiation pattern. I propose that the unexpected spatial dynamics of the early-replicating rDNA repeats serve to ensure streamlined efficient replication of the most heavily transcribed genomic loci while simultaneously reducing the risk of chromosome breaks and rDNA hyper-recombination.

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Section: Replicated Rdna Relocates To the Nucleolar Interior And Reasmentioning

confidence: 72%

Section: Replicated Rdna Relocates To the Nucleolar Interior And Reasmentioning

confidence: 99%

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

Dimitrova

2011

Journal of Cell Science

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“…In the rRNA gene region, the earliest labeled DNA fragments have been identified (8,21) and the growth and relative abundance of nascent DNA chains have been determined (21,29,60). These studies reveal two primary initiation sites: a 1-to 6-kb locus upstream of the rRNA gene promoter that is Ͼ10-fold more active than distal sites and a ϳ3-kb locus with weaker activity downstream of the 3Ј end of the gene.…”

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confidence: 97%

Identification of Primary Initiation Sites for DNA Replication in the Hamster Dihydrofolate Reductase Gene Initiation Zone

Kobayashi

Rein

DePamphilis

1998

Molecular and Cellular Biology

121

141

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Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-␤. The ratio of ϳ0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to -exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-␤ origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-␤). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-␤, ori-␤, and ori-␥), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.At least 22 replication origins have now been mapped in the chromosomes of flies, frogs, and mammals (references 14, 45, and 61 and references cited below) by using several different strategies (reviewed in reference 13). While all the data are consistent with bidirectional replication involving classical replication bubbles and forks, a complex and sometimes contradictory view of replication origins has emerged. While some studies conclude that most initiation events occur at specific sites analogous to those found in single-cell organisms such as yeast, Tetrahymena, and Physarum, other studies conclude that most initiation events are distributed throughout large DNA regions with no preference for one site over another. This paradox is best illustrated by studies on the mammalian rRNA and dihydrofolate reductase (DHFR) gene regions, where several different methods have been applied to the same genomic loci.When nascent DNA is labeled with nucleotide precursors during its biosynthesis, the data suggest that most initiation events occur at specific DNA sequences referred to as origins of bidirectional replication (OBRs). In the rRNA gene region, the earliest labeled DNA fragments have been identified (8, 21) and the growth and relative abundance of nascent DNA chains have been determined (21,29,60). These studies reveal two primary initiation sites: a 1-to 6-kb locus upstream of the rRNA gene promoter that is Ͼ10-fold more active than distal sites and a ϳ3-kb locus with weaker activity downstream of the 3Ј end of the gene. The major site appears to be conserved among species (37). In the DHFR gene region in Chinese hamster ovary (CHO) ce...

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“…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).…”

mentioning

confidence: 99%

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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Mapping of Replication Initiation Sites in Human Ribosomal DNA by Nascent-Strand Abundance Analysis

Cited by 64 publications

References 48 publications

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

Identification of Primary Initiation Sites for DNA Replication in the Hamster Dihydrofolate Reductase Gene Initiation Zone

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

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