Isolation and activity of Mcm4/6/7 complexes containing mutant proteins. A) Models for the subunit arrangement in a Mcm4/6/7 hexamer (left) and trimer (right) are depicted. The complexes are drawn viewing the C-terminal face of the ring and are based on known physical interactions between S. cerevisiae MCM proteins (modified from ). "SRF" indicates the conserved motif encoding the arginine finger. Other arrangements of the Mcm4/6/7 hexamer are formally possible, however these alternatives are less likely than the model shown given the current knowledge of Mcm and related protein structures and the known physical interactions between Mcm proteins [10,14,35,47,61]. B) Mcm4/6/7 complexes were reconstituted using all wild-type proteins ('WT'), Mcm4R701A, Mcm6 and Mcm7 ("4RA/6/7"); Mcm4, Mcm6R703A and Mcm7 ("4/6RA/7"); and Mcm4, Mcm6 and Mcm7R593A ("4/6/7RA") and the complexes were isolated by size exclusion chromatography as described in "Methods". The "WT" experiment shown here for comparison is from Figure 2. A portion of the indicated fractions was visualized by Coomassie Brilliant Blue stained SDS-PAGE (6%) to determine the peak fractions. The migrations of Mcm4, Mcm6 and Mcm7 (mutant or wild-type) through the gels are indicated to the right of each panel. The peak elution fractions of size standards analyzed separately are indicated below the bottom panel. C) DNA unwinding by wild-type and mutant Mcm4/6/7 complexes was compared. Each assay contained 750 ng of Mcm4/6/7. The numbers above the bars in the graph correspond to the ATP sites in part A. D) ATP hydrolysis was determined as described in "Methods". The mean rate per μg of Mcm4/6/7 is shown. The numbers above the bars in the graph correspond to the ATP sites shown in the hexamer model in part A and indicate which site is affected by the mutation.
Ma et al. BMC Biochemistry 2010 11:37 doi:10.1186/1471-2091-11-37