Live-cell imaging of endogenous CSB-mScarletI as a sensitive marker for DNA-damage-induced transcription stress

Transcription by RNA polymerase II (RNA Pol II) is crucial for cellular function, but DNA damage severely impedes this process. Thus far, transcription-blocking DNA lesions (TBLs) and their repair have been difficult to quantify in living cells. To overcome this, we generated, using CRISPR-Cas9-mediated gene editing, mScarletI-tagged Cockayne syndrome group B protein (CSB) and UV-stimulated scaffold protein A (UVSSA) knockin cells. These cells allowed us to study the binding dynamics of CSB and UVSSA to lesion-stalled RNA Pol II using fluorescence recovery after photobleaching (FRAP). We show that especially CSB mobility is a sensitive transcription stress marker at physiologically relevant DNA damage levels. Transcription-coupled nucleotide excision repair (TC-NER)-mediated repair can be assessed by studying CSB immobilization over time. Additionally, flow cytometry reveals the regulation of CSB protein levels by CRL4CSA-mediated ubiquitylation and deubiquitylation by USP7. This approach allows the sensitive detection of TBLs and their repair and the study of TC-NER complex assembly and stability in living cells. [Display omitted] •CRISPR-mediated, fluorescent tagging of endogenous TC-NER pathway proteins•CSB mobility determined by FRAP is a sensitive marker for transcriptional stress•CSB mobility measurements are a readout for TC-NER•Flow cytometry of labeled proteins enables study of abundance changes after DNA damage DNA damage that inhibits transcription and its repair has been thus far difficult to study in living cells. Therefore, using CRISPR-Cas9-mediated gene editing, we generated KI cells of the TC-NER proteins CSB and UVSSA. We show that measurement of especially CSB immobilization by fluorescence recovery after photobleaching (FRAP) is a sensitive marker to quantify transcription-blocking DNA damage and its repair. Zhou et al. generate cells with knockin fluorescent labeling of transcription-coupled repair proteins CSB and UVSSA. These tools enable fluorescence recovery after photobleaching (FRAP) studies to quantify transcription-blocking DNA damage and its repair in living cells.