A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus.

Tasheva, Elena S.; Roufa, D J

doi:10.1128/mcb.14.9.5628

Cited by 37 publications

(39 citation statements)

References 41 publications

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“…Six different methods for mapping replication origins reveal a primary initiation site for DNA replication in CHO cells that can be localized to a 2-kb region encompassing the original ori-␤ OBR identified by the transition between leading-and lagging-strand synthesis ( ) (6). (A) Leading-strand distribution (7,30); (B) earliest labeled DNA fragment (4,5,25,34,43,59); (C) nascent DNA strand length (53); (D) nascent DNA strand abundance in unsynchronized cells (47); (E) nascent DNA strand abundance in synchronized cells (this paper) (---, Br-DNA; --, RNA-DNA); (F) replication bubble trap (1); (G and H) Okazaki fragment distribution (6,52). Lightly shaded rectangles indicate the outer limits of the origin, while solid rectangles indicate the region of maximum origin activity.…”

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confidence: 99%

“…The strongest initiation site was coincident with the ori-␤ OBR in fragment FЈ originally identified in synchronized CHO cells by a transition from continuous to discontinuous DNA synthesis (6,7,30,52) and subsequently confirmed by nascent-strand length (53) and abundance (47) assays in exponentially proliferating CHO cells. A second, previously unrecognized initiation site (ori-␤Ј) of lower intensity was detected 5 kb further downstream in fragment F. Thus, when results from both the DHFR and rRNA gene regions are taken together, initiation zones in mammals appear to consist of one or more primary initiation sites (OBRs) as well as several low-frequency, secondary initiation sites that are detected only by 2D gel electrophoresis methods.…”

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confidence: 99%

“…The major site appears to be conserved among species (37). In the DHFR gene region in Chinese hamster ovary (CHO) cells, the earliest labeled DNA fragments have been identified (1,4,5,34,43), the polarity of replication forks has been determined by measuring both Okazaki fragment and leading-strand biases (6,7,30,52), and the growth and relative abundance of nascent DNA chains have been determined (47,53). These studies reveal two OBRs, a strong one (ori-␤) located within a 0.5-to 3-kb sequence ϳ17 kb downstream of the 3Ј end of the DHFR gene, and a weaker one (ori-␥) located about 23 kb further downstream.…”

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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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Section: Figmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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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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“…To determine whether or not a DMI could be detected at ori-␤ using the same strategy employed by Tasheva and Roufa (12), the methylation-sensitive restriction endonucleases (41) AluI (AGCT), AccI (GTMKAC), Alw26I (GGATC), or MslI (CAYN 4 RTG) were incubated individually with DNA from proliferating CHO cells isolated in our laboratory or with DNA samples provided by Tasheva and Roufa. All four of these enzymes cut within the putative DMI at ori-␤, and AluI and MslI cut within the putative DMI at ori-RPS14, another locus shown to contain an OBR (43). DNA products were amplified using PCR under conditions designed to give a linear response between the amount of template included in the reaction and the amount of product obtained (data not shown).…”

Section: The Putative Ori-␤ Dmi Was Not Detected By Four Methylation-mentioning

confidence: 99%

Absence of an Unusual “Densely Methylated Island” at the Hamster dhfr ori-β

Rein

Natale

Gärtner

et al. 1997

Journal of Biological Chemistry

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An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-␤ locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G 1 , S, or G 2 ؉ M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines ( m C) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of m C residues that complements the more commonly used hydrazine method. These methods were capable of detecting m C in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori-␤ DMI was not detected in any of these cell populations using any of these methods.DNA methylation at CpG dinucleotides in mammalian cells has been implicated as an important component of such pivotal processes as transcription (1), imprinting (2), recombination (3), carcinogenesis (4), development (5), and replication timing (6). CpG methylation is carried out by a methyltransferase that is associated with replication foci in the nucleus (7). In general, this enzyme methylates specifically hemimethylated CpG dinucleotides, although de novo methylation of unmethylated CpG dinucleotides can occur but at a low rate (8). In addition, this enzyme occasionally methylates cytosines in sequences other than CpG (9, 10). However, given the specificity of the mammalian methyltransferase, this non-target methylation should be of little significance in vivo because the pattern of non-CpG methylation will not be maintained during the subsequent round of DNA replication.Recently, an unusual form of DNA methylation has been implicated as an important component of mammalian origins of DNA replication. A densely methylated island (DMI) 1 has been reported at three different mammalian origins of DNA replication (11, 12). These DMIs were found only in proliferating cell populations and consisted of a 127-, 258-, or 516-bp stretch of DNA in which all cytosines were methylated, regardless of their sequence context (12). The discovery of DMIs has several important implications. First, this unprecedented methylation pattern suggested that a second (or modified) methylation enzyme exists in mammalian cells. Interestingly, limited proteolysis of the mamm...

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“…The human RPS14 locus encodes ribosomal protein S14 and is especially well suited for these studies. It is the only mammalian r-protein locus in which a drug selection system facilitates somatic genetics (Boersma et al 1979;Madjar et al 1982Madjar et al , 1983Diaz et al 1990;Diaz and Roufa 1992;Tasheva and Roufa 1993) and for which both genomic and eDNA clones carrying numerous wild-type and mutant alleles are available (Rhoads et al 1986;Rhoads and Roufa 1987;Diaz et al 1990;Overman et al 1993).…”

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Regulation of human RPS14 transcription by intronic antisense RNAs and ribosomal protein S14.

Tasheva

Roufa²

1995

Genes Dev.

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RNase protection studies reveal two stable RNAs (250 and 280 nucleotides) transcribed from the antisense strand of the human ribosomal protein gene RPS14's first intron. These transcripts, designated a-250 and a-280, map to overlapping segments of the intron's 5' sequence. Neither RNA encodes a polypeptide sequence, and both are expressed in all human cells and tissues examined. Although a-280 is detected among both the cells' nuclear and cytoplasmic RNAs, the great majority of o~-250 is found in the cytoplasmic subcellular compartment. As judged by its resistance to high concentrations of c~-amanitin, cell-free transcription of oL-250 and 0~-280 appears to involve RNA polymerase I. Tissue culture transfection and cell-free transcription experiments demonstrate that oL-250 and ot-280 stimulate S14 mRNA transcription, whereas free ribosomal protein S14 inhibits it. Electrophoretic mobility shift experiments indicate specific binary molecular interactions between r-protein S14, its message and the antisense RNAs. In light of these data, we propose a model for fine regulation of human RPS14 transcription that involves RPS14 intron 1 antisense RNAs as positive effectors and S14 protein as a negative effector.

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A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus.

Cited by 37 publications

References 41 publications

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

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

Absence of an Unusual “Densely Methylated Island” at the Hamster dhfr ori-β

Regulation of human RPS14 transcription by intronic antisense RNAs and ribosomal protein S14.

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