| Products/Services Used | Details | Operation |
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| Proteins, Expression, Isolation and Analysis | POLQ−/− HEK293T cells were generated by CRISPR-Cas9 engineering and were purchased from Genscript, Piscataway, NJ... Equal amounts (20 μg) whole-cell protein lysates were separated on 4–20% bis tris gels (GenScript) by electrophoresis then transferred onto Protran BA85 nitrocellulose membrane (Whatman, Germany) and immunoblotted with antibodies against Actin (MA-5-11869,1:20000, Invitrogen) or POLQ (PA5-69577,1:500, Invitrogen) overnight followed by secondary antibodies IRDye 800CW (926-32210, 1:10000) or IRDye 680CW (926-68073, 1:10000). Blots were scanned using ODYSSEY software. | Get A Quote |
DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in ad... More
