Gaubitz, C. | Search bioRxiv

bioRxiv2020

Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be actively opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader Replication Factor C (RFC) and sliding clamp PCNA are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryo-EM to an overall resolution of ~3.4 [A]. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a Limited Change/Induced Fit mechanism in which the clamp first opens, followed by DNA binding inducing opening of the loader to release auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

biochemistry↗

bioRxiv2019

A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry

Tailed bacteriophage use a DNA packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component acts as a molecular matchmaker by recognizing the viral genome as well as the main motor component, the large terminase (TerL). How TerS binds DNA and the TerL protein remains unclear. Here, we identify the TerS protein of the thermophilic bacteriophage P74-26. TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. Our cryo-EM structure shows that TerSP76-26 forms a ring with a wide central pore and radially arrayed helix-turn-helix (HTH) domains. These HTH domains, which are thought to bind DNA by wrapping the helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA binding domain imposes strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacks the conserved C-terminal {beta}-barrel domain used by other TerS proteins for binding TerL, suggesting that a well-ordered C-terminal {beta}-barrel domain is not necessary for TerS to carry out its function as a matchmaker.

biochemistry↗