Before a cell divides, it must make a perfect copy of its genetic blueprint through DNA replication, ensuring each new cell receives a complete set of instructions. The primary challenge is to copy every strand of DNA exactly once, as doing so incorrectly can lead to significant errors.
To manage this, cells use a control system known as replication licensing. This mechanism acts as a gatekeeper, granting permission for DNA to be copied at the right time and place. It ensures the integrity of the genome by preventing re-replication within a single cell division cycle.
The Pre-Replicative Complex
Replication licensing begins during the G1 phase, a preparatory period before DNA copying. The goal is to issue a “license” at specific DNA locations, marking them as approved starting points for replication. This is achieved by assembling proteins into a structure known as the Pre-Replicative Complex (Pre-RC).
First, the Origin Recognition Complex (ORC) identifies and binds to precise spots on the DNA known as “origins of replication.” These are the designated starting points for the process. Once in position, ORC acts as a docking site for another protein, Cdc6.
With ORC and Cdc6 bound, they recruit a third protein, Cdt1. Their primary job is to load the main component of the license onto the DNA: the Minichromosome Maintenance (MCM) complex. The MCM complex is a ring-shaped helicase that can unwind the DNA double helix.
The loading of the MCM complex is the final step in licensing the origin. The complete assembly of ORC, Cdc6, Cdt1, and the MCM ring forms the Pre-RC. The origin is now licensed, but the helicase remains inactive and the DNA wound, awaiting the signal to begin.
Activation of Replication Origins
Once licensed, an origin of replication remains dormant until the cell enters the S phase for DNA synthesis. The “go” signal is delivered by protein kinases. These enzymes act as molecular switches, turning on replication machinery by attaching a phosphate group to target proteins.
Two primary kinases trigger this activation: Cyclin-Dependent Kinases (CDKs) and Dbf4-dependent kinase (DDK). As the cell enters S phase, the activity of both rises sharply, providing the coordinated signal to begin DNA replication.
The signal is transmitted through phosphorylation, as CDKs and DDK add phosphate groups to Pre-RC components and other replication proteins. This modification changes the proteins’ shapes and interactions, initiating a cascade of events. This process ultimately activates the MCM helicase loaded during licensing.
With the MCM helicase active, it unwinds the DNA double helix at the origin, creating a replication “bubble.” This provides access for proteins like DNA polymerase to start synthesizing new DNA strands. The signal thus “fires” the origin, transitioning it from a licensed to an active state of replication.
Ensuring Once-Per-Cycle Replication
After an origin fires, the cell must ensure it cannot be licensed again until after cell division. The cell uses multiple strategies to dismantle the licensing system as S phase starts, destroying the “license” after one use.
High CDK activity also blocks the formation of new Pre-RCs. CDKs phosphorylate licensing factors, marking them for destruction or inactivation. For instance, the Cdc6 protein is tagged with phosphates and degraded, preventing a new MCM complex from being loaded.
Another control is the protein geminin, which inhibits the MCM loader Cdt1. Geminin levels are low during G1 but rise as the cell enters S phase. By binding to Cdt1, geminin blocks it from loading more MCM helicases, shutting down the licensing pathway.
As a final safeguard, MCM complex components are exported from the nucleus after replication is initiated. This physical separation prevents them from being reloaded onto the DNA. These mechanisms guarantee the licensing system is shut down once S phase begins, ensuring the genome is copied only once.
Replication Licensing and Disease
Failures in replication licensing can have severe consequences, including human diseases like cancer. Errors in copying DNA exactly once per cycle can lead to uncontrolled cell growth and genomic instability, which are hallmarks of cancer.
If re-licensing prevention fails, DNA regions can be copied multiple times in one cycle. This re-replication leads to gene over-amplification, which can transform a normal cell into a cancerous one. For example, overexpressing Cdt1 or Cdc6, or losing the inhibitor geminin, can cause the DNA damage that fuels tumor development.
Defects in the initial licensing machinery can also lead to developmental disorders. Since cell division is the basis of growth, replication problems can have significant effects. For instance, Meier-Gorlin syndrome, a rare disorder with developmental issues, is caused by mutations in genes for proteins in the ORC, Cdc6, or Cdt1.
These mutations impair the cell’s ability to license replication origins, leading to under-replication of the genome. This can slow cell proliferation and trigger cell death, disrupting normal growth and development. Accurate DNA licensing is therefore necessary for normal human health.