Figure 2

RecBD fusion polypeptides are shown in lanes 10–15 of part a and in lanes 8–13 of part b. Intact RecC polypeptides are shown in lanes 10–15 of part c and in lanes 4–9 of part d. RecBD fusion mutants do not assemble into the heterotrimer (in lanes 4–9 of part d). Extracts were from late log phase cultures of V2831 containing derivatives of plasmids pDWS2 (rec BCD⁺), pBD2728 and pBD2729. Fusion polypeptides in cell extracts were separated on denaturing (3–8% Tris acetate or 4–12% Bis-Tris buffer) and native (5% in 2 mM EDTA, 50 mM MOPS pH 7.0) polyacrylamide gels. Proteins were transferred to PVDF membrane and probed with mouse monoclonal antibodies specific for RecB, RecC and RecD, which were captured and visualized with horseradish peroxidase-linked antimouse IgG.