Fig. 2. Two distinct Holliday junction endonucleases. (A) Holliday junction resolution reactions contained either the branch migration-associated resolvase A or the Mus81-associated endonuclease. Nicked duplex products were analysed by neutral agarose gel electrophoresis. Reactions were carried out in the absence of ATP, and with the most highly purified fractions (0.5 µl) from MonoQ. (B) Detection of Mus81 by Western blotting using aliquots (20 µl) of the fractions used in (A). (C) Resolvase fractions from MonoQ were immunodepleted using anti-Mus81 or anti-GST antibodies coupled to protein A–Sepharose beads, as described in Materials and methods. The beads were then precipitated and the supernatants incubated with ³²P-labelled Holliday junction DNA. Lanes a and d, controls in which the resolvases were incubated on ice without Sepharose beads; lanes b and e, after pre-incubation with anti-GST–Sepharose beads; lanes c and f, after pre-incubation with anti-Mus81–Sepharose beads.