R-loop immunoprecipitation: a method to detect R-loop interacting factors
R-loops are non-B-DNA structures consisting of an RNA/DNA hybrid and a displaced single-stranded DNA. They arise during transcription and play important biological roles. However, perturbation of R-loop levels represents a source of DNA damage and genome instability resulting in human diseases, incl...
| Main Authors: | , |
|---|---|
| Format: | Book section |
| Language: | English |
| Published: |
Springer
2022
|
| _version_ | 1797108477737304064 |
|---|---|
| author | Beghè, C Gromak, N |
| author_facet | Beghè, C Gromak, N |
| author_sort | Beghè, C |
| collection | OXFORD |
| description | R-loops are non-B-DNA structures consisting of an RNA/DNA hybrid and a displaced single-stranded DNA. They arise during transcription and play important 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 with S9.6 antibody, specific for RNA/DNA hybrids, followed by Western blotting or mass spectrometry. Multiple specificity controls including addition of synthetic competitors and RNase H treatment are described to verify the specificity of identified R-loop-binding factors. The identification of new R-loop interacting factors and the characterization of their involvement in R-loop biology provides a powerful resource to study the role of these important structures in health and disease. |
| first_indexed | 2024-03-07T07:28:19Z |
| format | Book section |
| id | oxford-uuid:0de2032f-7040-4b51-83f1-4e3c8dba0624 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:28:19Z |
| publishDate | 2022 |
| publisher | Springer |
| record_format | dspace |
| spelling | oxford-uuid:0de2032f-7040-4b51-83f1-4e3c8dba06242022-12-22T09:35:26ZR-loop immunoprecipitation: a method to detect R-loop interacting factorsBook sectionhttp://purl.org/coar/resource_type/c_3248uuid:0de2032f-7040-4b51-83f1-4e3c8dba0624EnglishSymplectic ElementsSpringer2022Beghè, CGromak, NR-loops are non-B-DNA structures consisting of an RNA/DNA hybrid and a displaced single-stranded DNA. They arise during transcription and play important 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 with S9.6 antibody, specific for RNA/DNA hybrids, followed by Western blotting or mass spectrometry. Multiple specificity controls including addition of synthetic competitors and RNase H treatment are described to verify the specificity of identified R-loop-binding factors. The identification of new R-loop interacting factors and the characterization of their involvement in R-loop biology provides a powerful resource to study the role of these important structures in health and disease. |
| spellingShingle | Beghè, C Gromak, N R-loop immunoprecipitation: a method to detect R-loop interacting factors |
| title | R-loop immunoprecipitation: a method to detect R-loop interacting factors |
| title_full | R-loop immunoprecipitation: a method to detect R-loop interacting factors |
| title_fullStr | R-loop immunoprecipitation: a method to detect R-loop interacting factors |
| title_full_unstemmed | R-loop immunoprecipitation: a method to detect R-loop interacting factors |
| title_short | R-loop immunoprecipitation: a method to detect R-loop interacting factors |
| title_sort | r loop immunoprecipitation a method to detect r loop interacting factors |
| work_keys_str_mv | AT beghec rloopimmunoprecipitationamethodtodetectrloopinteractingfactors AT gromakn rloopimmunoprecipitationamethodtodetectrloopinteractingfactors |