Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA

Dobbs, Drena; Shaiu, Wen‐Ling; Benbow, Robert M.

doi:10.1093/nar/22.13.2479

Cited by 77 publications

(69 citation statements)

References 106 publications

(77 reference statements)

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“…Subsequent work has confirmed that replication starts in this location and at additional sites over a 12-kb region encompassing the c-myc gene (27,29,33,(38)(39)(40)(41). The 2.4-kb c-myc origin fragment contains a DNA unwinding element (DUE), which is a common feature of eucaryotic origins (11), a unique nucleosome organization, and three matches to the S. cerevisiae autonomously replicating sequence (ARS) consensus (39). Plasmids containing the 2.4-kb fragment of the c-myc origin replicate autonomously in vitro and when transfected into HeLa cells (3,16,27).…”

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confidence: 89%

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

Malott

Leffak

1999

Molecular and Cellular Biology

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DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.The bacterial replicon model of Jacob et al. (17) proposed that a trans-acting initiator protein binds to a cis-acting replicator element, defined as "a specific element of recognition upon which the corresponding initiator would act, allowing the replication of the DNA attached to the replicator." Consistent with this model, replication initiation in the simple eucaryote Saccharomyces cerevisiae is regulated by the interaction of cell cycle-dependent trans-acting factors with cis-acting DNA elements to establish replication bubbles and cause the initiation of DNA synthesis (6, 36). In metazoans, however, putative cis-acting replicator elements may be distributed over larger distances than the compact replicators of S. cerevisiae (4, 37), effect initiation at multiple sites, and comprise features of nuclear, chromatin, or DNA structure as well as DNA sequence (6,8,14,15). Biochemical assays suggest that DNA synthesis can initiate at multiple sites over regions as large as 55 kb in the hamster dihydrofolate reductase (DHFR) locus (9), the human c-myc locus (39, 41), and elsewhere (2, 7, 24, 35, 42). Replication does not initiate randomly across these large zones, although the degree to which replication initiates at preferred sites varies between replicons (13, 18, 20, 32). Nevertheless, the start sites f...

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confidence: 89%

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

Malott

Leffak

1999

Molecular and Cellular Biology

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“…The boundary locus 5'-TAAAAAAATA-Y is homologous to the consensus sequence found in Matrix Attachment Regions (MARs) and potential replication-initiation regions [10]. Alignment with five sequenced members of the HRS60 family shows (Fig.…”

Section: Resultsmentioning

confidence: 98%

The telomeric sequence is directly attached to the HRS60 subtelomeric tandem repeat in tobacco chromosomes

et al. 1995

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PCR and primers derived from the telomeric repeat (CCCTAAA)n and from the tobacco subtelomeric tandemly repetitive sequence HRS60 (EMBL X12489) were used to amplify the region linking the two loci. A 131 bp PCR product was obtained both from total tobacco DNA and from the DNA fraction enriched for telomeres. Its sequence only consists of the telomeric primer and the attached region of the HRS60 repetitive unit up to the end of the sequence complementary to the HRS60 primer. The site of direct continuity between the two sequences is formed by a (dA)7 tract.

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“…3) where the helical stability of the duplex, as measured by the ⌬G value, is at least 25 kcal/mol less than the average value for the whole sequence (95.9 kcal/mol). DUEs are known to be associated with eukaryotic replication origins, as its intrinsic helical instability facilitates replication initiation (8). DUEs are also a feature of S/MARs due to their property of stress-induced duplex destabilization, which is essential for S/MAR function (3).…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Mouse Recombination Hot Spot Locus Encoding a Novel Non-Protein-Coding RNA

Nishant

Ravishankar

Rao

2004

Molecular and Cellular Biology

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Our current knowledge of recombination hot spot activity in mammalian systems implicates a role for both the primary DNA sequence and the nature of the chromatin domain around it. In mice, the only recombination hot spots mapped to date have been confined to a cluster within the major histocompatibility complex (MHC) region. We present a high resolution analysis of a new recombination hot spot in the mouse genome which maps to mouse chromosome 8 C-D. Haplotype diversity analysis across 40 different strains of mice has enabled us to map recombination breakpoints to a 1-kb interval. This hot spot has a recombination intensity that is 10-to 100-fold above the genome average and has a mean gene conversion tract length of 371 bp. This meiotically active locus happens to be flanked by a transcribed region encoding a non-protein-coding RNA polymerase II transcript and the previously characterized repair site. Many of the primary DNA sequence features that have been reported for the mouse MHC hot spots are also shared by this hot spot locus and in addition, along with three other MHC hot spot loci, we show a new parallel feature of association of the crossover sites with the nuclear matrix.Most of the advances in our understanding of meiosis and recombination have come from studies of lower eukaryotes, in particular Saccharomyces cerevisiae. Recombination initiates via programmed double-strand breaks at the leptotene interval which are resected and undergo strand exchange and finally give rise to double Holliday junctions by the mid-pachytene interval. Mature crossover products follow around the end of pachytene (14,24). It is generally believed that a similar process operates in higher eukaryotes, including mammals. The rate of recombination is not a uniform function of the physical distance between two markers, leading to the concept of recombination hot spots and cold spots (36). In mice and humans, regions are generally termed as hot spots if their recombination frequency significantly exceeds 1 centimorgan (cM)/ Mb. There are three methods that have been used to map hot spots in mammals. The classical method is to correlate physical distances with genetic distances established by standard pedigree-based linkage analysis. The resolution of this method is limited by the density of markers, the number of individuals examined, and the number of recombination events that occur in a typical pedigree. Methods based on linkage disequilibrium (LD), which refers to the nonrandom association of alleles at linked sites, take into account ancestral recombination events, which would be far greater than those observed from any pedigree-based linkage study, leading to a finer mapping of recombination hot spots. Most measures of LD quantify the degree of association between pairs of markers which decreases as a function of physical distance between them due to increased probability of recombination between the markers. However, both of these techniques do not have the resolution to characterize crossovers at a molecular level. The ...

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Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA

Cited by 77 publications

References 106 publications

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

The telomeric sequence is directly attached to the HRS60 subtelomeric tandem repeat in tobacco chromosomes

Characterization of a Mouse Recombination Hot Spot Locus Encoding a Novel Non-Protein-Coding RNA

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