Protocol to measure end resection intermediates at sequence-specific DNA double-strand breaks by quantitative polymerase chain reaction using ER-AsiSI U2OS cells

Abstract

Summary: DNA end resection is a critical step in the homologous recombination pathway of repairing DNA double-strand breaks (DSBs) that can be visualized in cells by detecting the generation of single-stranded DNA (ssDNA) intermediates formed during the resection of the DSBs. Here, we describe quantitative polymerase-chain-reaction-based procedures to quantitatively measure ssDNA intermediates formed during the DNA end resection. Using the ER-AsiSI system, we use differential digestion patterns by restriction endonucleases that digest unresected double-stranded DNA at DSB sites.For complete details on the use and execution of this protocol, please refer to Fitieh et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Molecular Biology
• Protein Biochemistry