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Year:: 2015
Contributor:: Urban, John M. (creator); Gerbi, Susan A. (author)
Genre:: video recordings
Subject:: DNA replication; Origins of replication; Lambda exonuclease; Metazoans; human; Nucleosomes; Quadruplex nucleic acids; G-quadruplex
DOI: https://doi.org/10.7301/Z0SB43PC

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Abstract:: A popular method to map replication origins genome-wide is nascent strand sequencing (NS-seq) where short nascent strands with 5’ RNA primers are enriched by lambda exonuclease (λ-exo). Performing λ-exo digestion on sonicated DNA from G0 MCF7 cells (LexoG0), we found that λ-exo has difficulty digesting GC-rich sequences (consistent with single molecule studies) and G4-protected DNA (similar to Exo1). We used LexoG0 to control λ-exo digestion biases present in our NS-seq protocol, with validation at the rDNA locus. When controlling for λ-exo, G4 density no longer predicts origin density in the genome. Moreover, the majority of NS peaks did not overlap G4 motifs and the majority of G4 motifs did not overlap NS peaks. This suggests that G4s are not general determinants for origin specification, consistent with conclusions from orthogonal methods for mapping origins: bubble-trap, BrdU immunoprecipitation, and NS capture and release (Mesner et al. 2013, Mukhopadhyay et al. 2014; Bartholdy et al 2015; Kunnev et al. 2015). Nonetheless, we were interested in the possible role that G4s (or the G-rich nature of G4 motifs) might play at the subset (34.8%) of λ-exo controlled NS peak summits that had at least 1 G4 motif within 1 kb. Importantly, these G4 motifs were non-randomly distributed with respect to NS peak summits in an aggregate analysis, which revealed a wave-like G4 enrichment signal that increased after controlling λ-exo biases and was generally absent around LexoG0 peak summits. Interestingly, the periodicity of the waves was reminiscent of nucleosome spacing. To analyze the potential relationship of G4s with nucleosomes near origins, available nucleosome data from K562 and GM12878 cells was plotted around the G4-proximal subsets of summits in each dataset, which were all in regions of the genome with average and lower than average nucleosome enrichment. Nonetheless, the NS summits were flanked by nucleosomes from which the nucleosome signal spread out in a wave-like fashion with crest-to-crest distances typical of nucleosome spacing. Plotting the distribution of both G4s and nucleosomes together revealed that G4 enrichment crests were offset relative to nucleosome crests suggesting that a role of G4s near the G4-proximal subset of origins is in nucleosome positioning, which has been proposed as a more general role for G4s and G-rich sequences. More broadly, it is possible that a feature shared by all metazoan origins is a nearby sequence, one example of which are G4 motifs, that influences nucleosome positioning, which results in consistently available sites for opportunistic ORC binding, giving rise to apparent specificity in origin localization. For further reading on these topics see our 2015 Genome Research paper and our 2015 F1000 review of origin mapping methods in metazoans: Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins Michael S. Foulk, John M. Urban, Cinzia Casella and Susan A. Gerbi genome.cshlp.org/content/25/5/725 The hunt for origins of DNA replication in multicellular eukaryotes John M. Urban, Michael S. Foulk, Cinzia Casella and Susan A. Gerbi f1000.com/prime/reports/b/7/30/pdf

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Use and Reproduction: This work is licensed under a Creative Commons Attribution 4.0 International License

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Urban, John M., and Gerbi, Susan A., "G4 motifs and nucleosomes are phased around a subset of human replication origins" (2015). Brown University Open Data Collection. Brown Digital Repository. Brown University Library. https://doi.org/10.7301/Z0SB43PC

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Brown University Open Data Collection

This collection contains open and publicly-funded data sets created by Brown University faculty and student researchers. Increasingly, publishers, and funders are requiring that protocols, data sets, metadata, and code underlying published research be retained and preserved, their locations cited within …
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G4 motifs and nucleosomes are phased around a subset of human replication origins