13.2 Mechanisms of DNA replication

DNA Replication Process

DNA replication copies the entire genome before a cell divides, producing two identical DNA molecules from one original. Understanding how this works is central to cell biology because errors here can lead to mutations, disease, or cell death.

The process unfolds in three stages: initiation, elongation, and termination. Multiple enzymes coordinate at the replication fork to unwind the helix, synthesize new strands, and seal everything into continuous double-stranded molecules.

Steps of DNA replication

1. Initiation

   • Initiator proteins (e.g., DnaA in E. coli) recognize and bind the origin of replication (oriC), a specific AT-rich sequence that's easier to melt apart.
   • Helicase is loaded onto the DNA and unwinds the double helix by breaking hydrogen bonds between base pairs, creating a replication bubble with two replication forks moving in opposite directions.
   • Single-strand binding proteins (SSBs) stabilize the exposed single strands, preventing them from re-annealing or forming secondary structures.
   • Topoisomerase relieves the torsional strain (supercoiling) ahead of the replication fork that builds up as helicase unwinds the DNA.
   • Primase synthesizes short RNA primers (~10 nucleotides) complementary to each template strand, giving DNA polymerase a free 3'-OH group to extend from.

2. Elongation

   • DNA polymerase III binds at the primer-template junction and synthesizes new DNA in the 5' to 3' direction only.
   • The leading strand is synthesized continuously because it runs in the same direction as fork movement. It needs only a single RNA primer.
   • The lagging strand is synthesized discontinuously as short Okazaki fragments (~1,000–2,000 nt in prokaryotes, ~100–200 nt in eukaryotes). Each fragment requires its own RNA primer because the lagging template runs 3' to 5' relative to fork movement.
   • DNA polymerase I removes the RNA primers using its 5' to 3' exonuclease activity and replaces them with DNA nucleotides.
   • DNA ligase seals the remaining nicks (breaks in the sugar-phosphate backbone) between adjacent Okazaki fragments by catalyzing a phosphodiester bond.

3. Termination

   • Replication forks converge at a termination site (in prokaryotes, Tus proteins bound to ter sequences help stall the forks).
   • Any remaining gaps are filled and nicks are sealed by DNA polymerase I and DNA ligase.
   • The result is two complete daughter DNA molecules, each containing one original (parental) strand and one newly synthesized strand. This is semiconservative replication.

Key enzymes in replication

Notice that the original guide listed five enzymes. Topoisomerase and SSBs are just as essential at the fork. Topoisomerase prevents the DNA ahead of helicase from becoming impossibly overwound, and SSBs keep the separated strands accessible for copying.

DNA Replication Characteristics

Leading vs. lagging strands

The key constraint driving this distinction is that all DNA polymerases synthesize only in the 5' to 3' direction. Because the two template strands run antiparallel, the replication machinery handles them differently:

• Leading strand: The template runs 3' to 5' toward the fork, so the new strand grows 5' to 3' in the same direction as fork movement. Synthesis is continuous and requires only one primer.
• Lagging strand: The template runs 5' to 3' toward the fork, so the new strand must be built 5' to 3' away from the fork. Synthesis is discontinuous, producing Okazaki fragments that are later joined.

Role of Okazaki fragments

Okazaki fragments are the short DNA segments synthesized on the lagging strand. They exist because DNA polymerase can't synthesize toward the fork on that strand.

Each fragment goes through a lifecycle:

1. Primase lays down an RNA primer on the lagging-strand template.
2. DNA Pol III extends the primer, synthesizing DNA 5' to 3' until it reaches the primer of the previous fragment.
3. DNA Pol I removes the RNA primer and fills the gap with DNA.
4. DNA ligase seals the nick by forming a phosphodiester bond between the 3'-OH of one fragment and the 5'-phosphate of the next, creating a continuous sugar-phosphate backbone.

In prokaryotes, these fragments are roughly 1,000–2,000 nucleotides long. In eukaryotes, they're much shorter (100–200 nucleotides), partly because eukaryotic replication forks move more slowly.

Fidelity in DNA replication

Accurate copying is critical. Without error-correction mechanisms, DNA polymerase alone would introduce roughly one mistake per $10^{5}$ nucleotides. Multiple layers of correction bring the final error rate down to approximately one per $10^{10}$ nucleotides.

Layer 1: Base selection by DNA polymerase DNA Pol III selects the correct nucleotide based on Watson-Crick base pairing geometry. This alone provides an error rate of about $10^{-5}$.

Layer 2: Proofreading (3' to 5' exonuclease activity) If a wrong nucleotide is incorporated, the polymerase stalls because the mismatched base pair distorts the active site. The enzyme's built-in 3' to 5' exonuclease removes the incorrect nucleotide, and polymerization resumes. This improves accuracy by roughly 100-fold (to ~$10^{-7}$).

Layer 3: Mismatch repair (MMR) After replication, the mismatch repair system scans the newly synthesized DNA for errors that escaped proofreading. It distinguishes the new strand from the parental strand (in E. coli, by methylation status), excises the mismatched region on the new strand, and resynthesizes it correctly. MMR improves fidelity by another ~100–1,000-fold, achieving the final error rate of ~$10^{-10}$.

Together, these three layers ensure that the genome is copied with extraordinary precision, which is essential for maintaining genomic stability across generations of cell division.