The DNA damage response is essential for maintaining genome integrity. When DNA damage or replication stress occurs, a checkpoint signaling pathway is activated to arrest the cell cycle and initiate repair of the DNA damage, or trigger apoptosis. Now, researchers from Van Andel Institute (VAI) and the Rockefeller University have revealed how a key piece of the DNA repair process, called the 9-1-1 DNA checkpoint clamp, is enlisted to the site of DNA damage.
The findings are published in the journal Nature Structural and Molecular Biology in a paper titled, “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 researchers wrote. “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.”
This work illuminates new insights into the way cells ensure genetic instructions are properly passed from one generation of cells to the next. The project was led by the study’s co-corresponding authors Huilin Li, PhD, of VAI, and Michael E. O’Donnell, PhD, of the Rockefeller University and Howard Hughes Medical Institute.
“DNA damage can have severe consequences, including cancer and other diseases. Because of this, our cells have a host of checks and balances to ensure DNA integrity,” explained Li. “Our high-resolution structure of the 9-1-1 DNA checkpoint clamp as it interacts with the molecule that loads it onto the DNA strand gives us a detailed look at the essential process of DNA repair. We hope these insights can be leveraged toward the development of new therapeutic strategies for diseases linked to DNA damage.”
The structure was determined through use of VAI’s cryo-electron microscopes (cryo-EM). Using cryo-EM revealed that instead of loading onto DNA from the 3’ end like all other known DNA clamps, the 911 clamp is loaded onto DNA from the opposite end, called the 5’ end. This novel and unexpected finding reshapes what we know about DNA replication and sets the stage for further studies in this area.