CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin
Abstract
:1. Introduction
2. Results
2.1. Loss of CSB Leads to Unrestrained Fork Progression in Response to CPT at Low Concentrations
2.2. Loss of Fork Reversal Is Insufficient to Allow Restart of DNA Synthesis in CSB-KO Cells following 100 nM CPT-Induced Fork Stalling
2.3. CSB Is Epistatic to MUS81/RAD52/POLD3 to Restart DNA Replication in Response to a High Dose of CPT
2.4. Depletion of RAD51, BRCA1, or BRCA2 Restarts DNA Replication upon Treatment with 100 nM CPT in the Absence of CSB
2.5. Transcription Recovery Is Not Required for Restart of DNA Replication upon Treatment with 100 nM CPT in CSB-KO Cells Lacking RAD51, BRCA1, or BRCA2
2.6. PRIMPOL Mediates Restart of DNA Replication upon Treatment with 100 nM CPT in CSB-KO Cells Depleted for BRCA2 but Not RAD51 or BRCA1
2.7. LIG3 and LIG4, Respectively, Mediate Restart of DNA Replication upon Treatment with 100 nM CPT in RAD51- and BRCA1-Depleted CSB-KO Cells
2.8. CSB and BRCA2 Are a Toxic Combination to Genomic Stability and Cell Survival in Response to Treatment with 100 nM CPT
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Transfection
4.2. Small Interfering RNAs, Antibodies, and Drugs
4.3. DNA Fiber Assay
4.4. S1 Nuclease Assay
4.5. Transcriptional Recovery Assay (EU Incorporation)
4.6. Metaphase Chromosome Spreads
4.7. Immunoblotting
4.8. Clonogenic Survival Assays
4.9. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Batenburg, N.L.; Walker, J.R.; Zhu, X.-D. CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin. Int. J. Mol. Sci. 2023, 24, 12419. https://doi.org/10.3390/ijms241512419
Batenburg NL, Walker JR, Zhu X-D. CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin. International Journal of Molecular Sciences. 2023; 24(15):12419. https://doi.org/10.3390/ijms241512419
Chicago/Turabian StyleBatenburg, Nicole L., John R. Walker, and Xu-Dong Zhu. 2023. "CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin" International Journal of Molecular Sciences 24, no. 15: 12419. https://doi.org/10.3390/ijms241512419