Mcm4/6/7 binds single stranded DNA as a hexamer. Mcm4/6/7 was crosslinked in the presence of single stranded DNA using glutaraldehyde. The indicated amounts of Mcm4/6/7 were incubated with 20 nM 32P-dT60 and 1 mM ATP in 6 μl of 20 mM HEPES NaOH pH 7.5 and 10 mM magnesium acetate. After incubation at 25°C for 10 min, 0.4 μl of 2.3% glutaraldehyde was added. The mixture was then incubated at 37°C for 2 min before quenching the reaction with 0.8 μl 2 M Tris. After addition of 4 μl 60% glycerol, the samples were analyzed on a 5% polyacrylamide gel in 0.5 × TBE. A) A phosphorimager scan of crosslinking of Mcm4/6/7 to dT60 in the presence of ATP. The migration of size markers, based on Coomassie Brilliant Blue staining of the gel is indicated on the right. Note that in order to resolve trimer and hexamer sized, the gel was run so that free DNA migrated off the bottom of the gel. B) Crosslinking of Mcm4/6/7 in the presence and absence of DNA (as indicated). A Phosphorimage is shown on the right and the same gel, stained with GelCode Blue (Pierce) is on the left. Migration of size standards is shown on the right. C). Crosslinking of Mcm4/6/7 in the presence and absence of 1 mM ATP, as indicated. Migration of size standards is shown on the right. Lane 3 is an empty lane.
Ma et al. BMC Biochemistry 2010 11:37 doi:10.1186/1471-2091-11-37