DNA Replication

Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle.

The Steps:

• a portion of the double helix is unwound by a helicase.
• a molecule of DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix.
• because DNA synthesis can only occur 5' to 3', a second DNA polymerase molecule is used to bind to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase then stitches these together into the lagging strand.

Speed of Replication

Prokaryotes

The single molecule of DNA that is the E. coli genome contains 4.7 x 10⁶ nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. And thanks to the precision of the process (which includes a "proof-reading" function), the job is done with only about one incorrect nucleotide for every 10⁹ nucleotides inserted. In other words, more often than not, the E. coli genome (4.7 x 10⁶) is copied without error!

Eukaryotes

The average human chromosome contains 150 x 10⁶ nucleotide pairs which are copied at about 50 base pairs per second. The process would take a month (rather than the hour it actually does) but for the fact that there are many replication origins on the eukaryotic chromosome. Replication begins at some origins earlier in S phase than at others, but the process is completed for all by the end of S phase. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules.

Control of Replication

With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?

An observation: When a cell in G₂ of the cell cycle is fused with a cell in S phase, the DNA of the G₂ nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.

Two control mechanisms have been identified - one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.

Licensing: positive control of replication

• an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
• Accessory proteins called licensing factors. These accumulate in the nucleus during G₁ of the cell cycle. They include:
  • Cdc6 and Cdt1, which bind to the ORC and are essential for coating the DNA with
  • MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.
• Once replication begins in S phase,
  • Cdt1 and Cdc6 leave the ORCs
  • The MCM proteins leave in front of the advancing replication fork.

Geminin: negative control of replication

G₂ nuclei also contain at least one protein - called geminin - that prevents assembly of MCM proteins on freshly-synthesized DNA (probably by sequestering Cdt1).

As the cell completes mitosis, geminin is degraded so the DNA of the two daughter cells will be able to respond to licensing factors and be able to replicate their DNA at the next S phase.