DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp

The 9-1-1 DNA checkpoint clamp is loaded onto 5′-recessed DNA to activate the DNA damage checkpoint that arrests the cell cycle. The 9-1-1 clamp is a heterotrimeric ring that is loaded in Saccharomyces cerevisiae by Rad24-RFC (hRAD17-RFC), an alternate clamp loader in which Rad24 replaces Rfc1 in the RFC1-5 clamp loader of proliferating cell nuclear antigen (PCNA). The 9-1-1 clamp loading mechanism has been a mystery, because, unlike RFC, which loads PCNA onto a 3′-recessed junction, Rad24-RFC loads the 9-1-1 ring onto a 5′-recessed DNA junction. Here we report two cryo-EM structures of Rad24-RFC–DNA with a closed or 27-Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. Unlike RFC, which encircles DNA, Rad24 binds 5′-DNA on its surface, not inside the loader, and threads the 3′ ssDNA overhang into the 9-1-1 clamp from above the ring. Cryo-EM analysis reveals that the 9-1-1 clamp loader Rad24-RFC recognizes the 5′-recessed DNA end. The Rad24 subunit holds the DNA above the clamp, thereby loading the 9-1-1 clamp in the opposite direction of RFC loading DNA into the PCNA clamp.