Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells
Summary: It remains uncertain how global genome nucleotide excision repair (GG-NER) efficiently removes various helix distorting DNA lesions in the cell nucleus. Here, we present a protocol to assess the contribution of factors of interest to GG-NER using two types of fluorescence-microscopy-based t...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723003453 |
| _version_ | 1797796350898733056 |
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| author | Masayuki Kusakabe Kaoru Sugasawa |
| author_facet | Masayuki Kusakabe Kaoru Sugasawa |
| author_sort | Masayuki Kusakabe |
| collection | DOAJ |
| description | Summary: It remains uncertain how global genome nucleotide excision repair (GG-NER) efficiently removes various helix distorting DNA lesions in the cell nucleus. Here, we present a protocol to assess the contribution of factors of interest to GG-NER using two types of fluorescence-microscopy-based techniques. First, we describe steps for analyzing the localization of the factors upon local ultraviolet (UV) irradiation. We then detail the second technique, which quantifies the removal of UV-induced photolesions combined with lesion-specific antibodies and program-based image analysis.For complete details on the use and execution of this protocol, please refer to Kusakabe et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-03-13T03:31:45Z |
| format | Article |
| id | doaj.art-cd8b22204e3545aaad3991113f4d10f2 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-03-13T03:31:45Z |
| publishDate | 2023-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-cd8b22204e3545aaad3991113f4d10f22023-06-24T05:19:07ZengElsevierSTAR Protocols2666-16672023-09-0143102378Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cellsMasayuki Kusakabe0Kaoru Sugasawa1Biosignal Research Center and Graduate School of Science, Kobe University, Kobe 657-8501, JapanBiosignal Research Center and Graduate School of Science, Kobe University, Kobe 657-8501, Japan; Corresponding authorSummary: It remains uncertain how global genome nucleotide excision repair (GG-NER) efficiently removes various helix distorting DNA lesions in the cell nucleus. Here, we present a protocol to assess the contribution of factors of interest to GG-NER using two types of fluorescence-microscopy-based techniques. First, we describe steps for analyzing the localization of the factors upon local ultraviolet (UV) irradiation. We then detail the second technique, which quantifies the removal of UV-induced photolesions combined with lesion-specific antibodies and program-based image analysis.For complete details on the use and execution of this protocol, please refer to Kusakabe et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723003453Cell BiologyCell CultureMicroscopyMolecular Biology |
| spellingShingle | Masayuki Kusakabe Kaoru Sugasawa Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells STAR Protocols Cell Biology Cell Culture Microscopy Molecular Biology |
| title | Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells |
| title_full | Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells |
| title_fullStr | Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells |
| title_full_unstemmed | Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells |
| title_short | Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells |
| title_sort | fluorescence microscopy based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells |
| topic | Cell Biology Cell Culture Microscopy Molecular Biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723003453 |
| work_keys_str_mv | AT masayukikusakabe fluorescencemicroscopybasedassayassessingregulatorymechanismsofglobalgenomenucleotideexcisionrepairinculturedcells AT kaorusugasawa fluorescencemicroscopybasedassayassessingregulatorymechanismsofglobalgenomenucleotideexcisionrepairinculturedcells |