Titlebook: R-Loops; Methods and Protocol Andrés Aguilera,Alexey Ruzov Book 2022 The Editor(s) (if applicable) and The Author(s), under exclusive licen

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R-Loops and Mitochondrial DNA Metabolism,transcription and other aspects of DNA metabolism. The study of R-loops in mitochondria is in its infancy, and yet there is already clear evidence that they are predominantly located in the major regulatory region of the mammalian mitochondrial genome. Here, we describe how mitochondrial R-loops hav

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R-Loop Immunoprecipitation: A Method to Detect R-Loop Interacting Factors,nt biological roles. However, perturbation of R-loop levels represents a source of DNA damage and genome instability resulting in human diseases, including cancer and neurodegeneration. In this chapter, we describe a protocol which allows detection of R-loop interactors using affinity purification w

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,Studies on Protein–RNA:DNA Hybrid Interactions by Microscale Thermophoresis (MST),. MST uses localized temperature fields to measure the diffusion rates of the free and bound states of a fluorescently labeled protein, and to determine the dissociation constant . by fitting of the binding isotherm with a 1:1 binding model. Here, we describe the use of MST for quantitative analysis

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Immunostaining and Protein Pull-Down of Methyl-5-Cytosine-Marked RNA:DNA Hybrids,m.C) marked RNA:DNA hybrids containing the m.C modified RNA strand add another layer of regulation to the R-loop structure. Here we describe the detection of m.C modifications in the context of RNA:DNA hybrids using immunostaining, and identification of specific binding partners of m.C marked RNA:DN

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In Vitro Binding of GADD45A to RNA:DNA Hybrids,nstability, cells also harness RNA–DNA hybrids in scheduled, “regulatory,” R-loops to control gene expression. One regulatory role involves epigenetic gene regulation by the R-loop reader Growth Arrest and DNA Damage 45A (GADD45A). This small stress related protein promotes DNA demethylation by recr

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,Detection and Quantification of RNA Modifications on RNA–DNA Hybrids Using SID-UPLC-MS/MS,a protocol for quantitative analysis of RNA modifications on RNA–DNA hybrids using stable-isotope dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry (SID-UPLC-MS/MS). Supplemented by other techniques, this method can be instrumental in deciphering the roles of RNA modifications in R-loop metabolism.