Figure 3.

A Protein Attached to Fork DNA Can Hinder CMG Binding

(A) Schematic representation of experimental approach used in unwinding assays. “CMG→TA” refers to the strategy where CMG was allowed to bind the fork substrate before addition of traptavidin (TA). “TA→CMG” corresponds to CMG binding to the fork DNA that was pre-bound to TA.

(B and C) Unwinding of fork DNA bearing TA^Lag by (B) DmCMG or (C) ScCMG. Right panels show percentage of substrate unwound in each reaction. In all gel images, lane 1 corresponds to reaction lacking CMG, lane 2 to reaction lacking TA, lanes 3 and 4 to reactions including CMG and TA in different orders as indicated. All reactions included 5 nM DNA substrate and 50 nM helicase. DNA templates are Cy5 labeled at the 5′ end of the leading-strand template. Data represented here are mean ± SD from three independent experiments.

n.s., not significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S3.