TY - JOUR
T1 - Live-cell imaging of endogenous CSB-mScarletI as a sensitive marker for DNA-damage-induced transcription stress
AU - Zhou, Di
AU - Yu, Qing
AU - Janssens, Roel C.
AU - Marteijn, Jurgen A.
N1 - Publisher Copyright:
© 2023 The Author(s)
PY - 2024/1/22
Y1 - 2024/1/22
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85182757740&partnerID=8YFLogxK
U2 - 10.1016/j.crmeth.2023.100674
DO - 10.1016/j.crmeth.2023.100674
M3 - Article
C2 - 38176411
AN - SCOPUS:85182757740
SN - 2667-2375
VL - 4
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 1
M1 - 100674
ER -