Replication Initiation Point Mapping

Gerbi, Susan A.; Bielinsky, Anja‐Katrin

doi:10.1006/meth.1997.0526

Cited by 95 publications

(102 citation statements)

References 22 publications

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“…Purification of nascent-strand DNA Nascent-strand DNA was purified essentially as described elsewhere (Gerbi and Bielinsky 1997). Genomic DNA from 1-2 × 10 8 cells was isolated according to standard procedures and loaded onto a BND cellulose column, which isolates singlestranded DNA.…”

Section: Density Gradient Substitution Experimentsmentioning

confidence: 99%

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

Takeda¹,

Shibata²,

Parvin³

et al. 2005

Genes Dev.

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Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy. In order to duplicate the massive eukaryotic genome in a reasonable amount of time, DNA replication begins from multiple sites located throughout the genome called replication origins (for review, see Gilbert 2001). In the budding yeast, Saccharomyces cerevisiae, replication origins are defined by specific DNA sequences ∼100 base pairs (bp) in length. Plasmids containing these sequences replicate when transformed into cells and hence are referred to as autonomously replicating sequences, or ARS. Within each ARS is an 11-bp ARS consensus sequence (ACS) that is required for binding of the six-subunit origin recognition complex (ORC). ORC remains bound to chromatin throughout the cell cycle in S. cerevisiae, where it catalyzes the ordered assembly of replication factors that culminate in the loading of the replicative polymerases that begin DNA synthesis (Bell and Dutta 2002;Mendez and Stillman 2003). Although ORC and many of the proteins involved in replication are conserved in all eukaryotes, identification of conserved DNA sequences that define eukaryotic origins remains elusive (Gilbert 2004;Cvetic and Walter 2005). Similar plasmid transformation experiments that were successful in budding yeast have failed to identify ARS sequences in other eukaryotes. In mammalian cells, replication initiation occurs from poorly localized replication zones of 6-55 kb, although in a few cases the origin has been mapped to smaller lengths of DNA. Having welldefined origins in S. cerevisiae has greatly facilitated studying replication initiation. The initial discovery and characterization of ORC w...

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Section: Density Gradient Substitution Experimentsmentioning

confidence: 99%

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

Takeda¹,

Shibata²,

Parvin³

et al. 2005

Genes Dev.

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“…Two stringent methods have been used successfully to purify sequences located at replication initiation sites. One method relies on the trapping of bubble-shaped structures, and the other is based on isolation of transitory RNA-DNA SNS molecules (13,14). In the latter method, SNS between 1.5 kb and 2 kb can be purified specifically from an asynchronous population of cells because their RNA primers protect them from -exonuclease treatment, whereas broken genomic DNA is digested.…”

mentioning

confidence: 99%

“…Here we present a high-resolution map of replication origins in HeLa cells based on hybridization of short nascent strands (SNS) on DNA microarrays covering ENCODE regions. To construct this map we use one of the most stringent methods for isolating origins of replication based on the resistance of SNS to -exonuclease digestion (13). Because only small numbers of short nascent strands can be recovered, they must be amplified before hybridization to the microarray.…”

mentioning

confidence: 99%

Genome-wide studies highlight indirect links between human replication origins and gene regulation

Cadoret

Meisch

Hassan‐Zadeh

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

274

334

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To get insights into the regulation of replication initiation, we systematically mapped replication origins along 1% of the human genome in HeLa cells. We identified 283 origins, 10 times more than previously known. Origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no origins in large GC-poor regions. Origin sequences are evolutionarily conserved, and half of them map within or near CpG islands. Most of the origins overlap transcriptional regulatory elements, providing further evidence of a connection with gene regulation. Moreover, we identify c-JUN and c-FOS as important regulators of origin selection. Half of the identified replication initiation sites do not have an open chromatin configuration, showing the absence of a direct link with gene regulation. Replication timing analyses coupled with our origin mapping suggest that a relatively strict origintiming program regulates the replication of the human genome.chromatin structure ͉ DNA replication origin ͉ ENCODE regions ͉ genome-wide mapping ͉ CpG island C ontrolling the number of origins from which replication begins in a given chromosome is necessary to protect it from instability (1, 2). Mapping DNA replication starting points, known as ''origins of replication,'' would make a large contribution to understanding how genome replication is coordinated. Identification of a large number of replication origins is necessary to decipher the rules of origin specification. However, fewer than 30 origins have been identified in human cells (3), and they were mapped mostly in well-characterized transcribed regions, leaving gene-poor regions unexplored. The Encyclopedia of DNA Elements (ENCODE) project (4), launched to develop high-throughput methods for identifying functional elements, now provides a comprehensive view of gene expression and chromatin structure along 1% of the human genome (30 Mb, 44 regions) (5); it thus provides a powerful model for studying interactions among chromosome organization, gene regulation, and initiation of DNA replication. Origin selection is initiated by the binding of the origin recognition complex (ORC) to origin-proximal DNA sequences (6). In contrast to Saccharomyces cerevisiae, characterized metazoan origins do not conform to a clear consensus sequence, and the ORCs from higher eukaryotes exhibit no sequence specificity in vitro (7). Transcription factors at sites of replication initiation have been shown to stimulate replication in many systems, including viruses, yeast, Drosophila, and Xenopus (8, 9). This stimulation may be a consequence of direct interaction with components of the replication machinery or of facilitating the access of the replication complexes to DNA through recruitment of chromatin remodeling complexes (10-12). Here we present a high-resolution map of replication origins in HeLa cells based on hybridization of short nascent strands (SNS) on DNA microarrays covering ENCODE regions. To construct this map we use one of the most string...

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“…Choice of DNA polymerases-Although Vent (exo − ) DNA polymerase has been the polymerase of choice for RIP mapping [1][2][3] , this enzyme was not ideal under our experimental conditions. The possible reason for this is that the activity of this polymerase may not be compatible with betaine.…”

Section: Experimental Designmentioning

confidence: 99%

“…Using one-way PCR-based primer extension, Gerbi and Bielinsky successfully identified the precise transition point between continuous (leading strand) and discontinuous (lagging strand) DNA synthesis at the Saccharomyces cerevisiae ARS1 ori [2][3][4] . The original method developed by these authors 2 may be divided into the following six steps:…”

Section: Introductionmentioning

confidence: 99%

One-way PCR-based mapping of a replication initiation point (RIP)

Romero

Lee

2008

Nat Protoc

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The lamin B2 locus is the only mammalian origin whose replication initiation points (RIPs) have been mapped. Although this paper was published 8 years ago, no further mammalian RIP-mapping studies have been reported, largely due to technical difficulties of ligation-mediated (LM)-PCR used by the authors. Here, we report the development of a simple, one-way PCR-based protocol that allows one to accurately determine RIPs at mammalian origins. The procedure can be completed within 48 h from the time of cell lysis in the agarose gel. Nascent DNA is then isolated from the same gel after DNA is separated by alkaline gel electrophoresis. Subsequently, RIPs are determined by one-way PCR-based primer extension using labeled primers. Using this protocol, we have successfully mapped RIPs in the human DBF4 locus. As one-way PCR is routinely used by many scientists, this protocol will provide a powerful new tool for studying DNA replication in many organisms including mammalian cells.

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Replication Initiation Point Mapping

Cited by 95 publications

References 22 publications

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

Genome-wide studies highlight indirect links between human replication origins and gene regulation

One-way PCR-based mapping of a replication initiation point (RIP)

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