In preparation for loading (step 0), Rad24-RFC binds the 9-1-1 clamp to form a binary complex in the presence of ATP (or ATPS used in this study). In a mechanism similar to the mutual activation of RFC and PCNA, we suggest that binding energy between Rad24-RFC and 9-1-1 drives the DNA gate opening in Rad24-RFC (the A gate) and 9-1-1 (between Mec3 and Ddc1). This intermediate is grayed out as it is yet to be captured. In step 1, the shoulder DNA binds first to the Rad24-RFC external site. This is followed by the chamber DNA binding and passing through the 9-1-1 gate in steps 2 and 3. Once the DNA has fully entered, in steps 4 and 5, Mec3 moves toward Ddc1 to close the 9-1-1 gate, as indicated by the curved red arrow. The green Rad24 upper loop, with the help of the cyan Rfc5 plug, prevents DNA with a gap size shorter than 5 nt from entering the Rad24-RFC chamber. The consistent presence of four ATPS and one ADP throughout steps 1 to 4 suggests that ATP hydrolysis is not required for DNA binding and clamp gate closure. Stable DNA binding and clamp gate closure likely induce a conformational change in Rad24-RFC to trigger ATP hydrolysis, leading to the dissociation of Rad24-RFC from 9-1-1 in step 6, leaving 9-1-1 alone encircling the DNA 3' end.
