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

Abstract

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.

Keywords

• Cell Biology
• Cell Culture
• Microscopy
• Molecular Biology