Fig 14.

S. cerevisiae Slx1-Slx4 and Mus81-Mms4 do not interact, are independently nucleolytically active, and do not modulate the nuclease activity of one another. (A) The Mus81-Mms4 concentration was fixed at 5 nM, and Slx1-Slx4 was titrated either in the absence or in the presence of Mus81-Mms4 on four substrates: X12 (Holliday junction with branch point migratable over a 12-bp distance), RF-like, XO12 (Holliday junction with immobile branch point), and nXO12 (nicked Holliday junction). Reaction mixtures were incubated for 10 min at 30°C. Asterisks on joint molecules represent radiolabeling on respective oligonucleotides or, in the case of lanes, label the addition of 5 μl Slx1-Slx4 storage buffer. Arrows indicate approximate cleavage sites of both enzymes: for the replication fork-like substrate, cleavage could occur on either strand (Mus81-Mms4 upper strand, Slx1-Slx4 lower strand). (B) Quantitation of the results shown in panel A. (C) Tagged Slx4-13Myc and native Mus81 and Mms4 were immunoprecipitated from both untreated and MMS-, CPT-, and HU-treated cell extracts using antibodies against raised against Myc, Mus81, or Mms4 (WDHY2783). Immunoprecipitation using anti-Myc antibodies in an untagged strain served as a negative control. Precipitated proteins were separated by electrophoresis and subjected to sequential immunoblot analysis, using anti-Myc,-Mus81, and -Mms4 antibodies. Antibodies were stripped from the membrane prior to each subsequent blot.