Effect of RNAP roadblock on single molecule strand incision. Time-trace of incision assay carried out on the Rb-pREP4 construct using 2.5 nM MutS, 10 nM MutL, 10 nM MutH and RNA polymerase (RNAP) stalled between the bulge and the GATC site. RNAP addition ( 1 ) is followed by formation of a stalled elongation complex (sTEC), recognizable through its characteristic pattern of initial reduction in DNA extension followed by rapid partial increase (‘scrunching’). ( 2 ) Free components are washed out and ( 3 ) MMR components introduced. Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap. Red arrows indicate incision events. 