End-synapsis Assay

Abstract

Many environmental agents induce double-strand breaks (DSBs) in DNA. Unrepaired or improperly repaired DSBs can lead to cell death or cancer. Nonhomologous end joining is the primary DNA double-strand break repair pathway in eukaryotes. During NHEJ pathway, several proteins recognize and bind DNA ends, bring the ends in a synaptic complex and, finally, process and ligate the ends.Briefly, NHEJ starts with Ku protein. Ku binds the broken DNA ends and recruits the catalytic subunit of DNA dependent protein kinase (DNA-PKcs) forming DNA-PK. After processing, the XRCC4/Ligase IV complex executes the final ligation stimulated by Cernunnos-XLF. Here, we describe an end-synapsis assay. This assay can be used in order to delineate which proteins are necessary to bring the DNA ends in a stable synaptic complex during NHEJ. Briefly, NHEJ competent extracts from human cells were incubated with both a double-stranded DNA fragment bound to streptavidin-coated magnetic beads and the same soluble radio-labeled fragment. The beads were then washed in mild salt buffer and the radioactivity recovered with the beads was measured by scintillation counting. Control experiments without extracts or with DNA-free beads were run in parallel to determine unspecific background.