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. 2012 Jan 27;40(4):1868–1878. doi: 10.1093/nar/gks022

Figure 2.

Figure 2.

Bridging of DNA molecules by XRCC4–XLF. (A) Schematic of DNA-bridging assay. Proteins were incubated with magnetic beads linked to 1000-bp DNA and free 500-bp DNA. Beads were separated from supernatant and analyzed separately for presence of the 500-bp DNA. (B) an amount of 200 ng each of 1000- and 500-bp DNA fragments were incubated with XRCC4 (2 µM), XLF (2 µM) or DNA Ligase IV tandem BRCT domains (BRCTs, 2 µM). Top panel shows the analysis of the protein–DNA complexes in the supernatants. Bottom panel shows the recovery of DNA species on the beads. L = 1 kb DNA ladder (NEB). (C) Bridging assays performed as in (B) with mutants preventing XRCC4–XLF filament formation. (D) Bridging assays performed as in (B). XRCC41–157 and XLF1–224 are truncated proteins lacking C-terminal tails and DNA-binding activity.