The dimeric ring-shaped sliding clamp of E. coli DNA polymerase III (beta subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader gamma complex (homolog of eukaryotic Replication Factor C, RFC). The delta subunit of the gamma complex binds to the beta ring and opens it. The crystal structure of a beta:delta complex shows that delta, which is structurally related to the delta' and gamma subunits of the gamma complex, is a molecular wrench that induces or traps a conformational change in beta such that one of its dimer interfaces is destabilized. Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the beta ring opens spontaneously once a dimer interface is perturbed by the delta wrench.

The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol e below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle doublestranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.CMG helicase | DNA replication | DNA polymerase | origin initiation | replisome R eplicative helicases are hexameric rings in all domains of life (1-3). In bacteria and archaea, the replicative helicase is a homohexamer and encircles single-strand (ss) DNA at a replication fork. Some viral and phage replicative helicases are also ring-shaped hexamers, including bovine papilloma virus (BPV) E1, simian virus 40 (SV40) large T-antigen (T-Ag), and the T4 and T7 phage helicases. Unlike other replicative helicases, the eukaryotic replicative Mcm2-7 helicase is composed of six nonidentical but homologous Mcm subunits that become activated upon assembly with five accessory factors (Cdc45 and GINS tetramer) to form the 11-subunit CMG (Cdc45, Mcm2-7, GINS) (4-6). Numerous studies have outlined the process that forms CMG at origins in which the Mcm2-7 heterohexamer is loaded onto DNA as an inactive double hexamer in G1 phase, and becomes activated in S phase by several initiation proteins and cell-cycle kinases that assemble Cdc45 and GINS onto Mcm2-7 to form the active CMG helicases (7-9).Helicases assort into six superfamilies (SF1-SF6) based on sequence alignments (10). The SF1 and SF2 helicases are generally monomeric and the SF3-SF6 helicases are hexameric rings used in DNA replication and other processes. The bacterial SF4 and SF5 helicases contain RecA-based motors and translocate 5′-3′, whereas the eukarytic SF3 and SF6 helicases contain AAA+ (ATPases associated with diverse cellular activities)-based motors and translocate 3′-5′ (3, 10). Examples of well-studied hexameric helicases include t...

The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.