Fig 4.

Some nuclease-deficient mutants associate with cellular endonuclease activity in transfected cells. (A and D) SPA-tagged proteins were immunoprecipitated from transfected HeLa cells and visualized by immunoblotting with the rabbit anti-FLAG antibody followed by infrared detection. (B and C) Immunoprecipitates prepared as described for panel A were incubated with 50 ng linearized pUC19 (B) or 50 ng pEGFP-C1 (C) to visualize nuclease activity. Samples are labeled as indicated in panel A. Plasmid DNA incubated in the absence of immunoprecipitate (DNA only) is indicated as “-.” DNA was visualized using SYBR gold staining. (E) Immunoprecipitates from the experiment whose results are shown in panel D were incubated with 20 ng linearized pUC19 to visualize nuclease activity. Samples are labeled as indicated in panel D. Linearized pUC19 DNA incubated in the absence of immunoprecipitate (DNA only) is indicated as “-.” DNA was visualized using SYBR gold staining. (F) Immunoprecipitates from the experiment whose results are shown in panel D were also incubated with 50 ng circular pEGFP-C1, and the resulting nicked plasmid DNAs were radiolabeled using nick translation, separated by agarose gel electrophoresis, and visualized using a phosphorimager. Samples are labeled as indicated in panel D. pEGFP-C1 DNA incubated in the absence of immunoprecipitate (DNA only) is indicated as “-.” Circular pEGFP-C1 DNA incubated in the absence of immunoprecipitate and processed for nick translation without E. coli DNA polymerase I is indicated as “-Pol.”