Genome-scale identification of active DNA replication origins

Cayrou, Christelle; Grégoire, Damien; Coulombe, Philippe; Danis, Etienne; Méchali, Marcel

doi:10.1016/j.ymeth.2012.06.015

Cited by 42 publications

(42 citation statements)

References 28 publications

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“…In this approach, RNAprimed nascent DNA is first purified from replication origins, and then contaminated DNA is removed by lambda exonuclease (λ-Exo) digestion. We already described this method in detail (Cayrou et al 2011(Cayrou et al , 2012b and showed that it gives accurate and very reliable results, providing that the crucial λ-Exo digestion is performed in optimal conditions (Supplemental Figs. S1, S2; Cayrou et al 2011;Li et al 2014;Picard et al 2014).…”

Section: Origin Maps Reveal Three Different Classes Of Origins Definementioning

confidence: 99%

The chromatin environment shapes DNA replication origin organization and defines origin classes

et al. 2015

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To unveil the still-elusive nature of metazoan replication origins, we identified them genome-wide and at unprecedented high-resolution in mouse ES cells. This allowed initiation sites (IS) and initiation zones (IZ) to be differentiated. We then characterized their genetic signatures and organization and integrated these data with 43 chromatin marks and factors. Our results reveal that replication origins can be grouped into three main classes with distinct organization, chromatin environment, and sequence motifs. Class 1 contains relatively isolated, low-efficiency origins that are poor in epigenetic marks and are enriched in an asymmetric AC repeat at the initiation site. Late origins are mainly found in this class. Class 2 origins are particularly rich in enhancer elements. Class 3 origins are the most efficient and are associated with open chromatin and polycomb protein-enriched regions. The presence of Origin G-rich Repeated elements (OGRE) potentially forming G-quadruplexes (G4) was confirmed at most origins. These coincide with nucleosome-depleted regions located upstream of the initiation sites, which are associated with a labile nucleosome containing H3K64ac. These data demonstrate that specific chromatin landscapes and combinations of specific signatures regulate origin localization. They explain the frequently observed links between DNA replication and transcription. They also emphasize the plasticity of metazoan replication origins and suggest that in multicellular eukaryotes, the combination of distinct genetic features and chromatin configurations act in synergy to define and adapt the origin profile.

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Section: Origin Maps Reveal Three Different Classes Of Origins Definementioning

confidence: 99%

The chromatin environment shapes DNA replication origin organization and defines origin classes

et al. 2015

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“…The next step was enrichment of RNA-primed nascent strands by lambda exonuclease (Lexo) treatment, which eliminates single-stranded DNA with phosphorylated 5′-ends but preserves hybrid molecules containing 5′-terminal RNA moieties. This procedure is widely used in the study of replication initiation and is considered to be the most stringent method to isolate nascent DNA molecules that contain 5′-terminal RNA primers (26). Two cycles of T4 polynucleotide kinase (PNK) and Lexo treatment of the short singlestranded DNA fragments isolated from the sucrose gradient resulted in a 10-to 20-fold reduction in the amount of DNA, as estimated by quantitative real-time PCR (qPCR) with selected VACV primers.…”

Section: Resultsmentioning

confidence: 99%

“…After centrifugation at 800 × g for 3 min, the nuclei were discarded, and the virus factories were pelleted at 20,000 × g for 20 min. For further steps, the protocol described by Cayrou et al (26) was followed with some modifications. DNA was extracted from the pellets with DNAzol (Life Technologies) in the presence of proteinase K (200 μg/mL) and precipitated with ethanol.…”

Section: Methodsmentioning

confidence: 99%

“…However, the autonomous replication strategy to define origins was unsuccessful when applied to poxviruses: plasmids regardless of sequence efficiently replicate in poxvirus-infected cells, raising questions of whether specific origins are needed for the viral replication machinery (14)(15)(16). To investigate the mechanism of VACV replication, we exploited approaches recently proven effective for mapping eukaryotic replication origins by deep sequencing (26,32). However, in contrast to the conventional strategy, we sequenced predominantly Okazaki fragments instead of leading strands and used strand-specific deep sequencing.…”

Section: Consensus Nucleotides At the Start And End Of Vacv Dna Fragmmentioning

confidence: 99%

“…Additional advantages of deep sequencing included the ability to computationally discriminate viral and host DNA and the absence of a requirement for prior information on the distribution of replication origins. Lexo treatment was used to enrich DNA fragments with RNA primers, as previously described (26). The clear difference in the profiles of reads when Lexo treatment was omitted or if NaOH was used to remove RNA primers before Lexo indicated that VACV nascent chains start from RNA primers.…”

Section: Consensus Nucleotides At the Start And End Of Vacv Dna Fragmmentioning

confidence: 99%

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Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

Senkevich

Bruno

Martens

et al. 2015

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

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Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNAprimed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.vaccinia virus | DNA replication | DNA replication fork | DNA replication origin | Okazaki fragments P oxviruses comprise a large family of complex enveloped DNA viruses that infect vertebrates and insects and includes the agent responsible for human smallpox (1). In contrast to the nuclear location exploited for genome replication by many other DNA viruses, the 130-to 230-kbp linear double-stranded DNA genomes of poxviruses are synthesized within discrete, specialized regions of the cytoplasm known as virus factories or virosomes. Furthermore, most, if not all, proteins required for DNA replication are virus-encoded (2). Poxvirus genomes, as shown for the prototype species vaccinia virus (VACV), have covalently closed hairpin termini, so that the DNA forms a continuous polynucleotide chain (3). The hairpin is a 104-nucleotide (nt) A+T-rich incompletely base-paired structure that exists in two inverted and complementary forms. As expected for a genome with such covalently closed ends, VACV replicative intermediates are headto-head or tail-to-tail concatemers (4, 5). The concatemers exist only transiently because they are cleaved by a virus-encoded Holliday junction resolvase into unit length genomes with hairpin ends before incorporation into virus particles (6, 7). Because VACV genomes and concatemers resemble the replicative intermediates of the much smaller parvoviruses, the rolling hairpin model of replication originally proposed for the latter family was e...

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G4 motifs affect origin positioning and efficiency in two vertebrate replicators

Valton¹,

Hassan‐Zadeh²,

Lema³

et al. 2014

The EMBO Journal

195

196

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DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.

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Genome-scale identification of active DNA replication origins

Cited by 42 publications

References 28 publications

The chromatin environment shapes DNA replication origin organization and defines origin classes

The chromatin environment shapes DNA replication origin organization and defines origin classes

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

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