DNA replication, the duplication of a cell’s genetic material, is a fundamental process that must be executed with precision. To manage the enormous length of DNA, replication does not start randomly but at multiple specific points scattered across the chromosomes. “Origin firing” describes the tightly controlled molecular event that signals the beginning of the active DNA copying phase, known as the S phase of the cell cycle. This process requires the coordination of numerous proteins to ensure the entire genome is copied accurately before cell division.
Identifying the Starting Points for Replication
The starting location for DNA replication is called the origin of replication, and its selection is the first regulated step. In eukaryotic cells, this selection occurs during the G1 phase of the cell cycle in a process termed “licensing.” Licensing prepares the origin for potential firing by assembling the pre-Replication Complex (pre-RC).
The process begins when the Origin Recognition Complex (ORC) binds to the designated origin sequence on the DNA. ORC then acts as a scaffold to recruit co-loader proteins, including Cdc6 and Cdt1.
These co-loaders work together to load the core component of the replication machinery, the Mini-Chromosome Maintenance (MCM) complex. The MCM complex is a ring-shaped protein assembly that functions as the replicative helicase. It is loaded onto the double-stranded DNA as an inactive double hexamer.
Once the MCM helicase is loaded, the origin is considered “licensed” and competent to initiate replication. However, the helicase remains dormant, and no DNA unwinding occurs during the G1 phase.
The Molecular Steps of Origin Activation
Origin firing is the transition from the licensed, inactive MCM complex to the fully active, DNA-unwinding replication machine. This activation is triggered by the rising activity of two main classes of protein kinases: Cyclin-Dependent Kinases (CDK) and Dbf4-dependent kinase (DDK). These kinases become active as the cell transitions from G1 into S phase.
DDK acts first, directly phosphorylating specific subunits of the MCM helicase complex. This phosphorylation changes the MCM complex’s shape and activity, allowing it to recruit the key helicase-activating factor, Cdc45.
Following this, S-phase CDK activity promotes the recruitment of the GINS complex and other factors. The combination of MCM, Cdc45, and GINS forms the complete, active replicative helicase, known as the CMG complex.
The active CMG helicase then separates the two DNA strands, creating a replication bubble with two opposing replication forks. This unwinding exposes single-stranded DNA templates, allowing the subsequent recruitment of DNA polymerase and associated proteins. Synthesis of the new DNA strands then begins in a bidirectional manner away from the origin.
Ensuring Replication Happens Only Once
Genomic stability relies on the rule that a replication origin must fire only once per cell cycle. Re-replication would lead to catastrophic gene amplification and genome instability. The cell enforces this temporal control by creating an antagonistic relationship between the G1 phase and the S, G2, and M phases.
In G1, low kinase activity permits origin licensing; ORC is stable, and co-loaders like Cdc6 and Cdt1 are available to load the MCM helicase. Once the cell enters S phase, high CDK activity initiates firing but simultaneously prevents any new licensing events.
High CDK activity directly inhibits the formation of new pre-RCs through multiple mechanisms. For instance, CDK phosphorylates ORC components, reducing their ability to bind DNA. The co-loader Cdc6 is also phosphorylated by CDK, marking it for degradation by the proteasome machinery. Additionally, the co-loader Cdt1 is rapidly degraded during S phase through ubiquitination. By inactivating or degrading the proteins required for MCM loading, the cell ensures the MCM helicase cannot be reloaded onto the DNA until the high CDK activity drops in late mitosis and G1 phase.