Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells

Masayuki Kusakabe; Kaoru Sugasawa

doi:10.1016/j.xpro.2023.102378

Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells

STAR Protoc. 2023 Sep 15;4(3):102378. doi: 10.1016/j.xpro.2023.102378. Epub 2023 Jun 22.

Authors

Masayuki Kusakabe¹, Kaoru Sugasawa²

Affiliations

¹ Biosignal Research Center and Graduate School of Science, Kobe University, Kobe 657-8501, Japan.
² Biosignal Research Center and Graduate School of Science, Kobe University, Kobe 657-8501, Japan. Electronic address: ksugasawa@garnet.kobe-u.ac.jp.

Abstract

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.

Keywords: Cell Biology; Cell Culture; Microscopy; Molecular Biology.

MeSH terms

Cells, Cultured
DNA Damage* / genetics
DNA Repair
Excision Repair*
Microscopy
Nucleotides

Substances

Nucleotides