Alberto Riera

An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC–Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC–Cdc6 and Cdt1–MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC–Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC–Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC–Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated

A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2–7 (MCM2–7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2–7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2–7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.

In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2–7 hexamer dimerization

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2–7 onto DNA. Helicase loading involves two MCM2–7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2–7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC–Cdc6 interaction and MCM2–7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2–7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2–7. To determine whether Cdc6 regulates MCM2–7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2–7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2–7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2–7 recruitment, show that ATPase activity is required for MCM2–7 hexamer dimerization and demonstrate that MCM2–7 hexamers are recruited to origins in a consecutive process.

Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1

To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2–7 double hexamer onto DNA. Without ATP hydrolysis, ORC–Cdc6 recruits one Cdt1-bound Mcm2–7 hexamer, thus forming an ORC–Cdc6–Cdt1–Mcm2–7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2–7 winged-helix domains (WHDs) engage ORC–Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC–Cdc6 and Mcm2–7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2–7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2–7 hexamer by ORC–Cdc6.

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.

Helicase loading: How to build a MCM2-7 double-hexamer

A central step in eukaryotic initiation of DNA replication is the loading of the helicase at replication origins, misregulation of this reaction leads to DNA damage and genome instability. Here we discuss how the helicase becomes recruited to origins and loaded into a double-hexamer around double-stranded DNA. We specifically describe the individual steps in complex assembly and explain how this process is regulated to maintain genome stability. Structural analysis of the helicase loader and the helicase has provided key insights into the process of double-hexamer formation. A structural comparison of the bacterial and eukaryotic system suggests a mechanism of helicase loading.

From structure to mechanism—understanding initiation of DNA replication

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.