Fig. 3

Trypsinised DNA substrates and their cleavage by TDP1 and MUS81 complexes. DNA substrates bearing a small TOP1 peptide were prepared by treating TOP1-linked DNA with trypsin. A TDP1 removes the TOP1 peptide from nicked duplex, Y-form and 3′ flap. The trypsinised substrates were incubated with TDP1 at 30 °C for 15 min. The reaction was stopped with SDS and resolved by denaturing PAGE. Numbers under the gel pictures represent the percentage of cleavage product calculated from the sum of the modified substrate and product band intensities. B MUS81-EME1 (M-E1) cleaves trypsinised nicked duplex in contrast to Y-form DNA. C Mus81-Mms4 (M-M), as well as MUS81-EME1 (M-E1) and MUS81-EME2 (M-E2), efficiently cleave the trypsinised 3′ flap substrate (left). Quantification of the activity (right). Only the bands corresponding to the oligo 7⁻ bearing the small TOP1 peptide were quantified. The trypsinised substrates were incubated with increasing amounts of MUS81 complexes at 37 °C for 15 min. The reaction was stopped by adding SDS and resolved by denaturing PAGE