1. TRF2/RAP1complex mediates C‐NHEJ inhibition at telomeric ends through hindrance at the KU loading and DNA–PK activation steps.
TRF2/RAP1complex mediates C‐NHEJ inhibition at telomeric ends through hindrance at the KU loading and DNA–PK activation steps. (A) Western blotting analysis of HeLa protein extracts after immuno‐depletion as indicated. Protein samples were denatured and separated on 8% SDS–PAGE gel followed by electrotransfer on membrane and blotting with the antibodies as indicated. (B) EJ assay catalysed under standard reaction conditions with the indicated plasmids and HeLa extracts depleted as specified, in the presence or not of NU7026. DNA ligation products were separated by agarose gel electrophoresis followed by SYBR‐Green staining. (C) Quantification of independent experiments as shown in (B) (n=3). Relative ligation efficiency was calcultated as the % of ligation obtained in each case related to the ligation obtained on the control biopC plasmid with the control IgG‐depleted extracts. Error bars correspond to s.e.m. Black bars correspond to control IgG‐depleted extracts and grey bars to TRF2/RAP1‐depleted extracts. Absolute ligation efficiency was 4.6±0.8% s.e.m. for IgG‐depleted extracts on bioPC plasmid. (D) Pull‐down experiment under standard conditions with HeLa extracts mixed with the indicated biotinylated DNA fragments. Salt molarity during washing of the beads is specified. The initial amount of protein used during the pull‐down experiment was loaded as input. Protein samples were denatured and separated on 8% SDS–PAGE gel followed by electrotransfer on membrane and blotting with the antibodies as indicated. (E) Pull‐down experiment as in (D) but with immuno‐depleted HeLa extracts as indicated and washing under high salt conditions. C stands for the 502 bp non‐biotinylated control DNA fragment.
Oriane Bombarde et al. EMBO J. 2010;29:
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