Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic b...

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Veröffentlicht in:	The Journal of cell biology Jg. 220; H. 9
Hauptverfasser:	Crossley, Magdalena P, Brickner, Joshua R, Song, Chenlin, Zar, Su Mon Thin, Maw, Su S, Chédin, Frédéric, Tsai, Miaw-Sheue, Cimprich, Karlene A
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	United States 06.09.2021
Schlagworte:	Antibodies - chemistry Antibodies - metabolism BRCA1 Protein - antagonists & inhibitors BRCA1 Protein - genetics BRCA1 Protein - metabolism Cloning, Molecular DNA - chemistry DNA - metabolism DNA - ultrastructure DNA Helicases - antagonists & inhibitors DNA Helicases - genetics DNA Helicases - metabolism Escherichia coli - genetics Escherichia coli - metabolism Fluorescent Dyes - chemistry Fluorescent Dyes - metabolism Gene Expression Genes, Reporter Genetic Vectors - chemistry Genetic Vectors - metabolism Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism HeLa Cells Heterocyclic Compounds, 4 or More Rings - chemistry Heterocyclic Compounds, 4 or More Rings - metabolism Humans Inverted Repeat Sequences Multifunctional Enzymes - antagonists & inhibitors Multifunctional Enzymes - genetics Multifunctional Enzymes - metabolism Nucleic Acid Hybridization Optical Imaging - methods Protein Binding Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Ribonuclease H - genetics Ribonuclease H - metabolism RNA Helicases - antagonists & inhibitors RNA Helicases - genetics RNA Helicases - metabolism RNA, Double-Stranded - chemistry RNA, Double-Stranded - metabolism RNA, Double-Stranded - ultrastructure RNA, Small Interfering - genetics RNA, Small Interfering - metabolism Staining and Labeling - methods
ISSN:	1540-8140, 1540-8140
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Zusammenfassung:	R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.
Bibliographie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1540-8140 1540-8140
DOI:	10.1083/jcb.202101092