Mechanism of sliding clamp loading during DNA replication and repair in atomic detail.

Abstract

DNA replication is essential for all life. Efficient and fast replication is promoted by ring-shaped sliding clamp proteins. Moreover, the eukaryotic clamp, Proliferating Cellular Nuclear Antigen (PCNA) coordinates several other cellular pathways, including DNA repair. PCNA's role as the central hub for pathways that maintain genome stability makes its proper function important to human health. The main regulator of sliding clamp is the clamp loader AAA+ ATPase. The eukaryotic loader, Replication Factor C (RFC), opens the closed PCNA and loads it onto DNA. Despite years of study, it was unclear how RFC opens PCNA and subsequently binds DNA. To address this, we used single particle cryoEM to reconstruct multiple structures of the RFC:PCNA complex that delineate the steps in the clamp loading reaction, ranging from the first encounter complex to intermediates with PCNA loaded onto primer-template DNA without ATP hydrolysis. We observe that RFC opens PCNA with a ‘crab-claw-like’ motion that enables RFC to prefer PCNA binding before DNA. Furthermore, primer-template DNA, RFC's primary DNA substrate during replication, binds directly to the central chamber of the complex. However, this structure did not tell us how the complex binds to other DNA substrates, such as nicked or gapped DNA for DNA repair. Thus, we also determined structures of RFC:PCNA bound to those DNA and discovered a second DNA binding site. Both DNA binding sites contain ‘separation pins’ that melt DNA without ATP hydrolysis to provide the flexibility required for DNA binding. This is the first structural insight into how PCNA can accomplish its dual roles in DNA repair and replication, and is supported by our biochemical and cellular studies. In all, our study shows how a AAA+ ATPase can load sliding clamps at a variety of DNA architectures.