Mechanisms and regulation of DNA replication initiation in eukaryotes

Abstract

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Keywords: CMG; Cdc6; Cdt1; DNA replication; MCM2-7; ORC; helicase; initiator.

Figure 1

Mechanistic outline of DNA replication initiation in eukarya. During the G1 phase of the cell cycle, an origin-bound ORC•Cdc6 complex together with Cdt1 facilitates the sequential recruitment and loading of two MCM2-7 complexes into a stable double hexamer that encircles duplex DNA (pre-RC). At the onset of S phase, the helicase is activated, leading to origin unwinding. The recruitment of other initiation factors (Cdc45 and GINS, the pre-IC) and double-hexamer dissolution activate the helicase to drive fork progression as a single-stranded DNA-bound Cdc45•MCM2-7•GINS (CMG) complex. A color version of this figure is available online.

Figure 2

Molecular details of eukaryotic origins and mechanisms of ORC binding. (GREY) S. cerevisiae origins are distinctive among eukaryotes for conforming to a consensus sequence, the ACS. ScORC can bind the ACS directly and specifically, although interactions between the Orc1-BAH domain and nucleosomes can also modulate ORC origin selection. (PURPLE) Although they do not possess a strict consensus sequence, S. pombe origins are AT-rich. SpORC specifically binds such sites using a domain insertion unique to SpOrc4 that encodes a DNA-binding AT-hook motif. (GREEN) Metazoan ORC can be targeted to chromosomes through a variety of mechanisms, including the Orc1 BAH domain, the DNA-binding TFIIB domain of Orc6, and through interactions with chromatin-associated factors. A majority of metazoan origins are also predicted to contain G-quadruplex secondary structure elements, but how this feature affects ORC binding is currently unclear. A color version of this figure is available online.

Figure 3

ORC architecture. A) Cdc6/Orc homologs are characterized by three domains, two of which form the AAA+ module (green) and a third that encodes a winged-helix (WH) domain (grey). Bound nucleotide is shown as sticks (PDB = 1FNN). B) The D. melanogaster ORC heterohexamer is a crescent shaped molecule with the AAA+ (green surface) and WH (grey cartoon) domains forming a domain-swapped arrangement. Orc6 is bound by a domain insertion in the AAA+ domain of Orc3. Although the Orc1/Orc4 active site is required for activity, in the D. melanogaster structure Orc1 is disengaged from Orc4 and positioned above the plane of the AAA+ ring (PDB = 4XGC). A color version of this figure is available online.

Figure 4

MCM architecture. A) MCM homologs are characterized by three domains: NTD, AAA+, and CTD. The NTD can be subdivided into NTD-A (a small helical bundle), NTD-B (Zn-finger), and NTD-C (OB fold). The CTD forms a WH domain (for AAA+ and NTD, PDB = 3F9V; for WH domain, PDB = 2KLQ). B) Two physiologically relevant MCM oligomers have been observed, a hexamer that is formed by lateral interactions between the AAA+ and NTD domains of adjacent protomers, and a double hexamer that is formed by interactions between the NTD-B Zn-finger domains of two MCM2-7 rings. The double hexamer structure from S. cerevisiae Mcm2-7 is shown (Li et al., 2015), with one hexamer faded compared to the other. The inset shows a top-down view through the central cavity and the radial arrangement of eukaryotic MCM2-7 subunits (the double hexamer was built from two copies of PDB = 3JA8 fit to the EM density map EMD-6338 (Li et al., 2015)). A color version of this figure is available online.

Figure 5

Functional elements of MCM helicases. A) Each MCM monomer contains multiple functional elements, including DNA-binding/sensing motifs, regions that modulate ATPase activity, and loops that communicate between the NTD and AAA+ domain (PDB = 3JA8, chain 2). B) In the context of a hexamer, the MCM functional elements (excepting the external β-hairpin) line a central cavity through which DNA translocates (modeled after PDB = 3JA8, chains 4, 6, and 7). C) Symbol key. D) Detailed functional description for each MCM element known to contribute to activity. A color version of this figure is available online.

Figure 6

MCM2-7 complex loading and maturation into the CMG. Two sequential rounds of helicase recruitment and loading at origins is required for building an MCM2-7 double-hexamer. For both hexamers, DNA is threaded into the central channel through a discontinuity between MCM2 and MCM5. The first hexamer is recruited through direct interactions with the initiator (MCM3-Cdc6) and may require Cdt1 for overcoming an MCM6-mediated autoinhibited state of the helicase. After the first hexamer loads, both Cdc6 and Cdt1 are released. Rebinding of Cdc6 to ORC primes the system for recruiting and loading a second hexamer in the opposite direction as the first, an event that has been proposed to be controlled by ORC•Cdc6, but templated by the first MCM2-7 hexamer. Cdt1 and Cdc6 recruitment and ejection are required for both loading events. Phosphorylation of the double hexamer and other initiation factors by CDK and DDK facilitate origin melting, GINS and Cdc45 recruitment/assembly, DNA strand extrusion, and activation of the helicase for DNA unwinding. A color version of this figure is available online.

Figure 7

CMG helicase organization and dynamics. (A) MCM2-7 adopts a spiral, cracked-ring architecture with a discontinuity between MCM2 and MCM5. Upon incorporation into the CMG, Cdc45 and GINS convert the helicase into a planar form and seal off the MCM2/5 gate (EM density maps = EMD-1835 and EMD-1833 for MCM2-7 and CMG, respectively). (B) A view of the CMG from the AAA+ face illustrating the overall architecture of the complex (PDB = 3JC5 (Yuan et al., 2016)). (C) At least two conformational states exist for the CMG, a constricted state in which the AAA+ and NTD rings are coplanar (bottom panel), and a relaxed state where one end of the AAA+ tier lifts up from NTD tier (top panel). These conformations appear coupled to alterations in the relative disposition of the gating subunits, MCM2 and MCM5 (constricted and relaxed conformer PDB codes = 3JC5 and 3JC7, respectively). (D) The activated CMG helicase is thought to translocate along single-stranded DNA, unwinding downstream template through a combined steric exclusion and DNA wrapping mechanism. A color version of this figure is available online.

Figure 8

Mechanisms to prevent re-replication. Multiple, redundant mechanisms are utilized to prevent re-licensing of origins after S-phase has initiated. Whereas yeast seem to exclusively utilize CDK-dependent mechanisms to prevent re-licensing, metazoans also employ CDK-independent pathways for negatively regulating Cdt1 activity. A color version of this figure is available online.