14.4 DNA Replication in Prokaryotes

DNA Replication in Prokaryotes

DNA replication is how a prokaryotic cell copies its entire genome before dividing. Understanding this process is central to genetics because errors here can lead to mutations, and the enzymes involved show up repeatedly in molecular biology topics like PCR and genetic engineering.

Prokaryotic replication starts at a single origin of replication and moves outward in both directions. Three main phases drive the process: initiation, elongation, and termination. Each phase depends on a specific set of enzymes and proteins working in coordination.

DNA Structure Review

Before diving into replication, a quick refresher on DNA structure helps the steps make sense.

• DNA is made of nucleotides, each containing a deoxyribose sugar, a phosphate group, and a nitrogenous base.
• The two strands of the double helix are antiparallel, meaning one runs 5' to 3' while the other runs 3' to 5'. This has major consequences for how replication works on each strand.
• Replication follows the semiconservative model: each new double helix contains one original (template) strand and one newly synthesized strand. This was demonstrated by the Meselson-Stahl experiment.

Steps of Prokaryotic DNA Replication

1. Initiation

• Replication begins at a specific sequence called the origin of replication (oriC).
• Initiator proteins (DnaA in E. coli) recognize and bind to oriC, causing the double helix to separate locally.
• This opening creates two replication forks that will move in opposite directions, making replication bidirectional.

2. Elongation

This is where the bulk of DNA synthesis happens. Multiple enzymes coordinate at each replication fork:

1. DNA helicase unwinds the double helix ahead of the fork by breaking hydrogen bonds between base pairs.

2. Single-strand binding proteins (SSBs) coat the exposed single strands, preventing them from snapping back together or forming secondary structures like hairpins.

3. Topoisomerase (gyrase) works ahead of helicase to relieve the torsional strain that builds up as the helix unwinds. Without it, the DNA ahead of the fork would become impossibly overwound.

4. Primase synthesizes short RNA primers (about 8–12 nucleotides long) complementary to the template strand. These primers are necessary because DNA polymerase III cannot start a new strand from scratch; it can only add nucleotides to an existing 3' end.

5. DNA polymerase III extends from each primer, synthesizing new DNA in the 5' to 3' direction only. This creates an asymmetry at the fork:

   • Leading strand: The template runs 3' to 5', so Pol III can follow the fork continuously with a single primer.
   • Lagging strand: The template runs 5' to 3', so Pol III must work away from the fork in short segments called Okazaki fragments (roughly 1,000–2,000 nucleotides each). Each fragment needs its own RNA primer.

6. DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides, filling the gaps.

7. DNA ligase seals the remaining nicks by forming phosphodiester bonds between adjacent Okazaki fragments, producing a continuous strand.

3. Termination

• The two replication forks eventually meet at the termination region on the opposite side of the circular chromosome.
• Specific termination (Ter) sequences and Tus proteins help stall the forks so they don't overshoot.
• Topoisomerases resolve any remaining supercoiling or interlinking of the two daughter chromosomes so they can be separated during cell division.

Key Enzymes in DNA Replication

Why DNA Polymerase Needs a Primer

This is a point that trips students up. DNA polymerase III is powerful, but it has a limitation: it can only add nucleotides to an existing strand. It cannot place the very first nucleotide on a bare template. That's why primase must lay down a short RNA primer first. Once that 3'-OH end exists, Pol III takes over. Later, Pol I swaps the RNA for DNA so the final product is pure DNA.

Leading vs. Lagging Strand

The antiparallel nature of DNA forces replication to work differently on each strand:

• Leading strand: Synthesized continuously toward the fork. Only one primer is needed. This is the simpler side.
• Lagging strand: Synthesized in pieces (Okazaki fragments) moving away from the fork. Each fragment requires a separate primer, and DNA ligase must stitch them together afterward.