Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging.
| Autor: | Crossley MP; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA., Brickner JR; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA., Song C; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA., Zar SMT; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA., Maw SS; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA., Chédin F; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA., Tsai MS; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA., Cimprich KA; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA. |
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| Jazyk: | angličtina |
| Zdroj: | The Journal of cell biology [J Cell Biol] 2021 Sep 06; Vol. 220 (9). Date of Electronic Publication: 2021 Jul 07. |
| DOI: | 10.1083/jcb.202101092 |
| Abstrakt: | 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. (© 2021 Crossley et al.) |
| Databáze: | MEDLINE |
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