Figure 2. The juxtaposition of DNA ends and an ssDNA gap triggers activation of the DNA damage signal. (A) Nuclear extracts were incubated for the indicated time periods with open circular plasmid DNA (lanes 1–5), linear duplex DNA (lanes 6–10), gap circular DNA (lanes 11–15), or gapped linear duplex DNA (lanes 16–20). The indicated proteins were analyzed by Western blotting. (B) ATR activation depends on accessible DNA ends. (lanes 1–3) dsDNA and gDNA were biotinylated at both ends and incubated with nuclear extracts. (lanes 4–6) Biotinylated DNA substrates (5 nM) were preincubated with 30 nM streptavidin for 15 min at 37°C before addition of nuclear extracts. (lanes 7–9) Unmodified DNA substrates were preincubated with streptavidin before addition of nuclear extracts. The DNA substrates are represented schematically. Biotin (black circles) and streptavidin (gray circles) are shown.
Figure 2.

The juxtaposition of DNA ends and an ssDNA gap triggers activation of the DNA damage signal. (A) Nuclear extracts were incubated for the indicated time periods with open circular plasmid DNA (lanes 1–5), linear duplex DNA (lanes 6–10), gap circular DNA (lanes 11–15), or gapped linear duplex DNA (lanes 16–20). The indicated proteins were analyzed by Western blotting. (B) ATR activation depends on accessible DNA ends. (lanes 1–3) dsDNA and gDNA were biotinylated at both ends and incubated with nuclear extracts. (lanes 4–6) Biotinylated DNA substrates (5 nM) were preincubated with 30 nM streptavidin for 15 min at 37°C before addition of nuclear extracts. (lanes 7–9) Unmodified DNA substrates were preincubated with streptavidin before addition of nuclear extracts. The DNA substrates are represented schematically. Biotin (black circles) and streptavidin (gray circles) are shown.

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