Journal: Nucleic Acids Research
Article Title: Efficient processing of abasic sites by bacterial nonhomologous end-joining Ku proteins
Figure Lengend Snippet: Bsu Ku binds circular DNA. ( A ) Left panel , Bsu Ku binding to covalently closed pUC19 plasmid. The assay was performed as described in Materials and Methods incubating the indicated amounts of Bsu Ku with 100 ng of supercoiled plasmid pUC19. Products were analysed on 0.7% agarose electrophoresis. One half of each reaction mixture was loaded directly onto the agarose gel (direct loading lanes), while the other half was being treated with Proteinase K. After proteolytic digestion, those samples were loaded in the same agarose gel (Proteinase K treatment lanes). The electrophoretical mobility of the pUC19 plasmid in each case is indicated. In lane c, DNA was incubated with 50 ng of BSA. Right panel , Bsu Ku binding to supercoiled and relaxed plasmid DNA. The assay was performed essentially as described above. ( B ) Bsu Ku AP lyase activity can act on circular substrates. The assay was performed as described in Materials and Methods, incubating either 142 nM of Bsu Ku with 100 ng of plasmid without AP sites (pcDNA3.1) or 18, 36, 72 and 142 nM of Bsu Ku with 100 ng of plasmid containing AP sites (pcDNA3.1-AP) (left panel). The absence and presence of the AP sites in pcDNA3.1 and pcDNA3.1-AP, respectively, was confirmed after digestion with h APE1. As a control of the linear form, plasmids were also digested with EcoRI. NC : nicked circles; SC : supercoiled; L : linear. The assay shown in the right panel was performed by incubating 100 ng of plasmid without (pUC19) or with (pUC19-AP) AP sites with either h APE1 or 142 nM of Bsu Ku, as indicated. Figure 5B , right panel is a composite image made from different parts of the same experiment.
Article Snippet: Agarose gel retardation Two micrograms of supercoiled pUC19 plasmid (Fermentas) were incubated with 10 U of the nickase Nt.BStNBI to obtain nicked pUC19.
Techniques: Binding Assay, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Incubation, Activity Assay, Activated Clotting Time Assay