Figure 8

DNA binding properties of a non-core RAG1 domain. (A) Increasing concentrations ranging from 0-3.0 μM of MBP-CND were titrated into radiolabeled ds DNA substrate containing the RSS. (B) Increasing concentrations, ranging from 0-5.0 μM, of tagless CND were titrated into radiolabeled ds WT 12-RSS. Due to difficulty in resolving the protein:DNA complex, the amount of unbound radiolabeled WT 12-RSS was quantified for each data point. Data presented is representative of three independent experiments. (C) Increasing concentrations of either WT 12-RSS or mutant nonamer (MN) 12-RSS competed with radiolabeled WT 12-RSS for binding to MBP-CND. For quantification of the competition experiments, the amount of radiolabeled WT 12-RSS bound to CND in the absence of competitor was set at 1.0 and subsequent data are represented as a function of this initial value. Data presented is representative of at least three separate experiments. (D) EMSA analysis to compare complex formations between MBP-CND and three separate radiolabeled DNA substrates, which each consist of the sequence corresponding to the 16 nt coding flank of the WT 12-RSS substrate (see Methods). Lanes 1-3 contain the ds 16 bp coding flank, lanes 4-6 contain the coding flank as a fully complementary 32 nt hp substrate, and lane 7 contains the ss 16 nt top strand coding flank sequence. The concentrations of MBP-CND and the percentage of DNA substrate bound are shown below each lane. In panels (A) and (D), protein-DNA complexes were resolved on a 3.5/8% discontinuous, non-denaturing gel.