دورية أكاديمية
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Ultra-deep Coverage Single-molecule R-loop Footprinting Reveals Principles of R-loop Formation.

التفاصيل البيبلوغرافية
العنوان: Ultra-deep Coverage Single-molecule R-loop Footprinting Reveals Principles of R-loop Formation.
المؤلفون: Malig M; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA 95616, USA; Integrative Genetics and Genomics Graduate Group, University of California Davis, Davis, CA 95616, USA., Hartono SR; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA 95616, USA., Giafaglione JM; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA 95616, USA., Sanz LA; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA 95616, USA., Chedin F; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA 95616, USA. Electronic address: flchedin@ucdavis.edu.
المصدر: Journal of molecular biology [J Mol Biol] 2020 Mar 27; Vol. 432 (7), pp. 2271-2288. Date of Electronic Publication: 2020 Feb 24.
نوع المنشور: Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.
اللغة: English
بيانات الدورية: Publisher: Elsevier Country of Publication: Netherlands NLM ID: 2985088R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1089-8638 (Electronic) Linking ISSN: 00222836 NLM ISO Abbreviation: J Mol Biol Subsets: MEDLINE
أسماء مطبوعة: Publication: Amsterdam : Elsevier
Original Publication: 1959- : London : Academic Press
مواضيع طبية MeSH: R-Loop Structures* , Transcription, Genetic*, DNA/*chemistry , Embryonal Carcinoma Stem Cells/*metabolism , RNA/*chemistry , Single-Cell Analysis/*methods, Embryonal Carcinoma Stem Cells/cytology ; Humans
مستخلص: R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP) approaches based on the anti-DNA:RNA hybrid S9.6 antibody revealed that R-loops form dynamically over conserved genic hotspots. We have developed an orthogonal approach that queries R-loops via the presence of long stretches of single-stranded DNA on their looped-out strand. Nondenaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long-read, single-molecule PacBio sequencing allows the identification of R-loop 'footprints' at near nucleotide resolution in a strand-specific manner on long single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) revealed a strong agreement between S9.6-based and bisulfite-based R-loop mapping and confirmed that R-loops form over genic hotspots, including gene bodies and terminal gene regions. Based on the largest single-molecule R-loop dataset to date, we show that individual R-loops form nonrandomly, defining discrete sets of overlapping molecular clusters that pileup through larger R-loop zones. R-loops most often map to intronic regions and their individual start and stop positions do not match with intron-exon boundaries, reinforcing the model that they form cotranscriptionally from unspliced transcripts. SMRF-seq further established that R-loop distribution patterns are not simply driven by intrinsic DNA sequence features but most likely also reflect DNA topological constraints. Overall, DRIP-based and SMRF-based approaches independently provide a complementary and congruent view of R-loop distribution, consolidating our understanding of the principles underlying R-loop formation.
(Copyright © 2020 Elsevier Ltd. All rights reserved.)
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معلومات مُعتمدة: R01 GM120607 United States GM NIGMS NIH HHS; T32 GM008799 United States GM NIGMS NIH HHS
فهرسة مساهمة: Keywords: DNA:RNA immunoprecipitation; Nondenaturing bisulfite conversion; R-loops; S9.6 antibody; SMRT-sequencing
المشرفين على المادة: 63231-63-0 (RNA)
9007-49-2 (DNA)
تواريخ الأحداث: Date Created: 20200228 Date Completed: 20200827 Latest Revision: 20210327
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC7669280
DOI: 10.1016/j.jmb.2020.02.014
PMID: 32105733
قاعدة البيانات: MEDLINE
الوصف
تدمد:1089-8638
DOI:10.1016/j.jmb.2020.02.014