Journal:
Article Title: The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase ? and increases its DNA substrate utilisation efficiency: implications for DNA repair
doi: 10.1093/nar/gkh652
Figure Lengend Snippet: DNA polymerase β physically interacts with the 9-1-1 complex. (A) Sf9 cell extracts (45 μg) expressing the 9-1-1 complex were incubated with His-tagged pol β (3 μg). From these extracts, anti-Rad9 immunoprecipitation was performed by using a polyclonal anti-Rad9 antibody. (B) GST-pulldown experiments were performed in the presence of either GST or GST–pol β (5 μg) and purified 9-1-1 complex (1.8 μg). (C) GST-pulldown experiments were performed in the presence of either GST or GST–pol β (5 μg) and Sf9 cell lysates expressing either Rad1 or Hus1 separately (15 μg). (D) Sf9 cell extracts (15 μg) expressing only Rad9 were incubated with purified His-tagged pol β (3 μg). Anti-Rad9 immunoprecipitation was performed by using a polyclonal anti-Rad9 antibody. The presence of coprecipitated proteins was determined by SDS–PAGE followed by western blot analysis. (E) Purified His-tagged pol β (2 μg) and 9-1-1 complex (6 μg) were incubated together and the proteins were subsequently separated on a Sephacryl S-200 column as described in Materials and Methods. The presence of pol β in the eluted fractions was detected by determining DNA polymerase activity as described in Material and Methods. The presence of each of the 9-1-1 complex proteins in the corresponding fractions was determined by SDS–PAGE followed by western blot analysis. His-tagged pol β (2 μg) and the 9-1-1 complex (6 μg) alone were analyzed as controls separately on a Sephacryl S-200 column. The incorporations of [3H]dTTP into acid-precipitable material is represented as broken line (for free pol β) and dotted line (for free 9-1-1 to exclude DNA polymerase contamination).
Article Snippet: The goat anti-Rad1 antibody (N-18) was from Santa Cruz biotechnology.
Techniques: Expressing, Incubation, Immunoprecipitation, Purification, SDS Page, Western Blot, Activity Assay
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![DNA polymerase β physically interacts with the 9-1-1 complex. (A) Sf9 cell extracts (45 μg) expressing the 9-1-1 complex were incubated with His-tagged pol β (3 μg). From these extracts, anti-Rad9 immunoprecipitation was performed by using a polyclonal anti-Rad9 antibody. (B) GST-pulldown experiments were performed in the presence of either GST or GST–pol β (5 μg) and purified 9-1-1 complex (1.8 μg). (C) GST-pulldown experiments were performed in the presence of either GST or GST–pol β (5 μg) and Sf9 cell lysates expressing either <t>Rad1</t> or Hus1 separately (15 μg). (D) Sf9 cell extracts (15 μg) expressing only Rad9 were incubated with purified His-tagged pol β (3 μg). Anti-Rad9 immunoprecipitation was performed by using a polyclonal anti-Rad9 antibody. The presence of coprecipitated proteins was determined by SDS–PAGE followed by western blot analysis. (E) Purified His-tagged pol β (2 μg) and 9-1-1 complex (6 μg) were incubated together and the proteins were subsequently separated on a Sephacryl S-200 column as described in Materials and Methods. The presence of pol β in the eluted fractions was detected by determining DNA polymerase activity as described in Material and Methods. The presence of each of the 9-1-1 complex proteins in the corresponding fractions was determined by SDS–PAGE followed by western blot analysis. His-tagged pol β (2 μg) and the 9-1-1 complex (6 μg) alone were analyzed as controls separately on a Sephacryl S-200 column. The incorporations of [3H]dTTP into acid-precipitable material is represented as broken line (for free pol β) and dotted line (for free 9-1-1 to exclude DNA polymerase contamination).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3528/pmc00443528/pmc00443528__gkh652f1.jpg)