Journal: Journal of Biological Chemistry

Article Title: Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

doi: 10.1074/jbc.m611125200

Figure Lengend Snippet: FIGURE 7. The DNA binding activity of Ku at RAG bound signal ends. A, a signal end complex is defined here as RAG1, RAG2, and HMG1 bound to two- oligonucleotide DNA duplexes, one containing the 12-RS and the other con- taining the 23-RS. A streptavidin-biotin complex was used to block the ends of the DNA duplexes distal to the site of cleavage. B, approximately 10 ng/l of purified RAG1 and RAG2 and 425 nM HMG1 were incubated with 0.4 nM radiolabeled23-RS-containingand10nM12-RS-containingDNAfragmentsat 37 °C for 10 min. 5 M streptavidin was added at 25 °C for 5 min prior to addition of 25 nM Ku. Antibody supershifts required an additional 10-min room temperature incubation step with either 0.2 g of the monoclonal anti- body to Ku or 1 l of a polyclonal antisera recognizing the maltose binding domain fused to recombinant RAG proteins. The inferred composition of each of the generated species is indicated to the side of the panel: species I, 23-RS; species II, HMG1-bound 23-RS; species III, RAGs and HMG1 bound to the 23-RS; species IV, SEC; species V, streptavidin-blocked SEC (upper arrow indi- cates -MBP supershift); species VI, Ku bound to incompletely formed SEC (upper arrow indicates -Ku supershift).

Article Snippet: The relative amounts of Ku and histoneH3 in each of the excised complexes were determined by semi- quantitative Western analysis probing with a polyclonal rabbit antibody raised against native, recombinant human Ku and a polyclonal antibody against histone H3 (Ab1791; Abcam) using fluorescent detection and a Typhoon imager (GE Healthcare).

Techniques: Binding Assay, Activity Assay, Blocking Assay, Purification, Incubation, Recombinant, Generated