Helicase loading at chromosomal origins of replication

Abstract

Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.

Figure 1.

Helicase loading at the E. coli replication origin (oriC) is a stepwise process. Near the top of the figure, DNA sequence elements in E. coli oriC are shown (see Leonard and Méchali 2013 for details). Binding sites for Fis and IHF are shown, which are believed to alter the architecture of oriC, and their roles are described in detail in Leonard and Méchali (2013). The four domains of DnaA shown in green are represented by the overlaid numbers. In step 1, DnaA complexed to ATP binds to the DnaA boxes, I-, τ-, and C-sites to form a DnaA oligomer. Following the unwinding of the region of oriC containing the 13-mers, DnaA then loads a DnaB–DnaC complex on each of the separated strands (step 2). In step 3, primase (DnaG) interacts with the amino-terminal region of DnaB. Primer formation by primase (steps 4 and 5) leads to the dissociation of DnaC from the carboxy-terminal domain of DnaB, which is necessary to activate DnaB as a DNA helicase (see Bell and Botchan 2013). Although not shown, the primers displayed in black are then extended by the cellular replicase, DNA polymerase III holoenzyme, for the continuous synthesis of each leading strand at each replication fork (see Johansson and Dixon 2013). A dimer of this DNA polymerase is believed to be at each replication fork. DnaB is proposed to interact with one unit of a dimer of DNA polymerase III holoenzyme, coordinating the unwinding of the parental DNA with DNA synthesis. As this DNA helicase translocates in the 5′-to-3′ direction to support replication fork movement, primase occasionally interacts with DnaB to synthesize additional primers. These primers are used by the unit of the DNA polymerase dimer that synthesizes Okazaki fragments.

Figure 2.

Structure of the Mcm2–7 complex. (A) The Mcm2–7 proteins assemble into a hexameric toroid. Each Mcm2–7 complex includes one copy of the Mcm2–7 proteins that are arranged in the indicated order around a central channel. (B) Directionality of Mcm2–7 movement. The Mcm2–7 proteins move in a 3′ → 5′ direction along ssDNA. By analogy with the archaea homologs, the carboxy-terminal AAA⁺ motif is proximal to the replication fork.

Figure 3.

Model for eukaryotic replicative DNA helicase loading. After localization to the origin DNA, ATP-bound ORC recruits Cdc6 bound to ATP. The resulting ORC–Cdc6 complex then recruits two Cdt1/Mcm2–7 complexes via interactions between Cdt1 and Orc6. Although this illustration suggests that the Mcm2–7 complexes have initiated interactions at their amino termini at this stage, it is also possible that these interactions only occur during or after helicase loading. The interactions between ORC, Cdc6, Cdt1, and Mcm2–7 are proposed to result in the opening of the Mcm2–7 ring at the Mcm2/5 gate. Cdc6 ATP hydrolysis results in the loading of an Mcm2–7 double hexamer around double-stranded origin DNA and the release of Cdt1. Whether the DNA enters the Mcm2–7 central channel before (upon initial ring opening) or after (as illustrated) Cdc6 ATP hydrolysis is unknown. ORC ATP hydrolysis is proposed to lead to the release of Cdc6–ADP and loaded Mcm2–7 from ORC. ORC ADP/ATP exchange leads to resetting of the loading machinery, allowing a new round of helicase loading to initiate.