Figure 8. Mot1-mediated displacement of TBP and NC2 from end-blocked DNA templates.
Electrophoretic mobility shift analysis of ATP-dependent disruption of Mot1:TBP:DNA complexes with and without NC2 as in Figure 2, but using DNA substrates that carry a digoxygenin label at one end (circle) and fluorescein for DNA detection at the other end (star). Reactions in lanes 13 and 14 were performed using DNA alone without digoxygenin modification, and demonstrate that the addition of digoxygenin antibody (Y-shape) had no effect on the mobility of unmodified DNA. Blocking of either end by the antibody did not result in a detectable decrease in TBP dissociation activity, suggesting that Mot1 does not translocate an intact TBP:NC2 clamp along DNA but locally disrupts the TBP:DNA:NC2 complex.