bsihkai  (New England Biolabs)

Journal: bioRxiv

Article Title: Break-induced replication is activated to repair R-loop-associated double-strand breaks in SETX-deficient cells

doi: 10.1101/2024.06.29.601361

Figure Lengend Snippet: A. WT and SETX -KO U2OS cells were treated with 10 Gy IR. Cell lysates were collected at indicated time points and checked with western blotting. B. Illustration of site-specific end resection assay in EGFP-BIR-5085 reporter. Cells are infected with I-SceI-expressing lentivirus to induce DSBs. After two days, genomic DNA (gDNA) is extracted and digested with indicated restriction enzyme (RE): BsiHKAI for the left DSB ends, ApoI for the right DSB ends, or SacII as mock for both ends. RE-resistant ssDNA is quantified by qPCR using primer sets around the RE recognition sites. C. WT and SETX -KO U2OS cells were infected with lentivirus expressing RPA2-EGFP to make constant expression of RPA2-EGFP. Cells with RPA2-EGFP expression were treated with 30 mW laser marked in black lines to induce DNA damage. The formation of RPA2-EGFP strips was recorded at the indicated time points. D. U2OS EGFP-BIR-5085 reporter cells with Flag-RNASEH1(D210N) expression were infected with SETX shRNA or vector. Four days later, cells were harvested for ChIP analysis. E. U2OS EGFP-BIR-5085 reporter cells with or without RNASEH1-V5 expression were infected with SETX shRNA or vector. Three days after infection, cells were infected with I-SceI-expressing lentivirus to induce DSBs. Three days after the second infection, cells were harvested for gDNA for end resection assay. F. WT and SETX -KO U2OS cells with or without RNASEH1-V5 expression were treated with 10 Gy IR. Cell lysates were collected one hour after IR and checked with western blotting.

Article Snippet: 1 ug genomic DNA was digested separately with NEB restriction enzyme BsiHKAI (left DSB end), ApoI (right DSB end) or SacII (mock) overnight.

Techniques: Western Blot, Resection Assay, Infection, Expressing, shRNA, Plasmid Preparation