Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader

Abstract

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC’s switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair

“…Because the 14 Å gap in PCNA is narrower than the 20 Å width of a dsDNA, and 3′-DNA1 is already inside the RFC central chamber, this is likely a post-entry intermediate in which the DNA gate has partially closed. These open and closed PCNA states resemble the open and closed clamp states observed in the T4 phage clamp-clamp loader complex ( Kelch et al, 2011 ) and the recent RFC−PCNA−DNA structure ( Gaubitz et al, 2022 ; Figure 1—figure supplement 4c ).…”

“…Moreover, clamp loaders of bacterial and eukaryotic systems only need ATP binding to crack open the clamp ring; neither ATP hydrolysis nor DNA is required ( Hingorani and O’Donnell, 1998 ; Kelch et al, 2012b ; Kelch et al, 2012a ; Turner et al, 1999 ; Zhuang et al, 2006 ). DNA binding appears to properly engage the ATPase sites for hydrolysis, leading to closure of the clamp around the duplex and dissociation of the clamp loader from DNA ( Anderson et al, 2009 ; Chen et al, 2009 ; Gaubitz et al, 2022 ; Kelch et al, 2012b ; Liu et al, 2017 ; Marzahn et al, 2015 ; Sakato et al, 2012a ; Trakselis et al, 2003 ).…”

“…A recent cryo-EM study captured yeast RFC in a series of intermediate states during loading of the PCNA clamp onto DNA, including an intermediate in which the PCNA ring is open by 20 Å, sufficiently wide for dsDNA to pass through ( Gaubitz et al, 2022 ). This conformation was not observed in the crystallographic study of the T4 phage clamp−loader−DNA complex ( Kelch et al, 2011 ).…”

Distinct Motifs in ATAD5 C-Terminal Domain Modulate PCNA Unloading Process

“…During PCNA loading by RFC, PCNA first interacts with RFC1 and multiple interactions between PCNA and RFC subunits occur during the opening of the PCNA ring [ 21 ]. It is possible that the ATAD5-PCNA interaction dynamically changes during the PCNA-unloading process.…”

“…Recent studies on RFC-PCNA interactions during PCNA loading provide insight into how ATAD5-RLC opens PCNA during the unloading [ 21 ]. Similar to RFC, ATAD5 forms a ring-shaped pentameric complex with RFC2-5.…”