Fig 3.
Sedimentation analysis of Mus81-Mms4. (A) Overlay of four sedimentation experiments in which the relative levels of heterodimer in the fractions were determined by anti-Mms4 immunoblots. Five to 10 μg (His)10Mus81-Mms4 was separated on 5 to 20% sucrose gradients. Fraction numbers were converted to sedimentation coefficient values (S value) by interpolation from standard curves generated by standard proteins present in the gradients. Each data set was calibrated using its own intragradient standard curve. Gradients were made with GF buffer containing 150 mM NaCl and no magnesium, except for the filled circles gradient, which contained 3 mM Mg(OAc)2, and the open squares gradient, which contained 500 mM NaCl. (B) Activity assays of a representative gradient fractionation (triangles in panel A). Two and a half microliters of each fraction was mixed with 7.5 μl of a cocktail to adjust the final mix to 150 mM NaCl, 3 mM Mg(OAc)2, and the indicated substrate concentrations. In the first lane, 2.5 μl of GF buffer was substituted as a negative control. The peak fraction contains 32 nM Mus81-Mms4, as determined by quantitative immunoblotting. HJ cleavage by Mus81-Mms4 requires excess protein over substrate, as previously found (2, 3). (C) Representative sedimentation standard curve used for S-value interpolation.