@article {Zheng2021.10.01.462756, author = {Fengwei Zheng and Roxana E. Georgescu and Nina Y. Yao and Michael E. O{\textquoteright}Donnell and Huilin Li}, title = {Rad24-RFC loads the 9-1-1 clamp by inserting DNA from the top of a wide-open ring, opposite the mechanism of RFC/PCNA}, elocation-id = {2021.10.01.462756}, year = {2021}, doi = {10.1101/2021.10.01.462756}, publisher = {Cold Spring Harbor Laboratory}, abstract = {In response to DNA damage, the ring-shaped 9-1-1 clamp is loaded onto 5{\textquoteright} recessed DNA to arrest the cell cycle and activate the DNA damage checkpoint. The 9-1-1 clamp is a heterotrimeric ring that is loaded in S. cerevisiae by Rad24-RFC, an alternative clamp loader in which Rad24 replaces the Rfc1 subunit in the RFC1-5 clamp loader of PCNA. Unlike RFC that loads the PCNA ring onto a 3{\textquoteright}-ss/ds DNA junction, Rad24-RFC loads the 9-1-1 ring onto a 5{\textquoteright}-ss/ds DNA junction, a consequence of DNA damage. The underlying 9-1-1 clamp loading mechanism has been a mystery. Here we report two 3.2-{\r A} cryo-EM structures of Rad24-RFC bound to DNA and either a closed or 27 {\r A} open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. The Rad24 subunit specifically recognizes the 5{\textquoteright}-DNA junction and holds ds DNA outside the clamp loader and above the plane of the 9-1-1 ring, rather than holding DNA inside and below the clamp as in RFC. The 3{\textquoteright} ssDNA overhang is required to obtain the structure, and thus confers a second DNA binding site. The bipartite DNA binding by Rad24-RFC suggests that ssDNA may be flipped into the open 9-1-1 ring, similar to ORC-Cdc6 that loads the Mcm2-7 ring on DNA. We propose that entry of ssDNA through the 9-1-1 ring triggers the ATP hydrolysis and release of the Rad24-RFC. The key DNA binding residues are conserved in higher eukaryotes, and thus the 9-1-1 clamp loading mechanism likely generalizes.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2021/10/01/2021.10.01.462756}, eprint = {https://www.biorxiv.org/content/early/2021/10/01/2021.10.01.462756.full.pdf}, journal = {bioRxiv} }