11.2 DNA Replication

DNA replication is the process cells use to copy their entire genome before dividing. Understanding how this works at the molecular level is central to microbial genetics, since errors or variations in replication drive mutation, evolution, and gene transfer in microbes.

This section covers the replication model, the enzymes involved, how leading and lagging strands differ, and how bacteria and eukaryotes handle replication differently.

DNA Replication Models and Processes

Semiconservative model of DNA replication

Before replication was well understood, scientists proposed three competing models for how DNA copies itself. The Meselson-Stahl experiment (1958) confirmed the semiconservative model, which works like this:

• The two strands of the parent DNA molecule separate.
• Each parent strand acts as a template for building a new complementary strand.
• The result is two daughter DNA molecules, each containing one original parent strand and one newly synthesized strand.

The name "semiconservative" makes sense once you see it: each daughter molecule conserves half of the original parent DNA. This is distinct from the conservative model (where the entire parent molecule would stay intact) or the dispersive model (where old and new DNA would be mixed throughout both strands).

Bidirectional nature of DNA replication

Replication doesn't proceed in just one direction from the starting point. It moves both ways simultaneously from the origin of replication (oriC in bacteria), creating two replication forks that travel in opposite directions. This effectively doubles the speed of the process.

At each replication fork, the two template strands pose different challenges because DNA strands are antiparallel (one runs 5'→3' while the other runs 3'→5'), yet DNA polymerase can only synthesize in the 5'→3' direction:

• Leading strand: Synthesized continuously in the 5'→3' direction, following the replication fork as it opens. DNA polymerase can work without interruption here.
• Lagging strand: Synthesized discontinuously as short fragments (Okazaki fragments) because the polymerase must work away from the replication fork to maintain 5'→3' synthesis. Each time the fork opens further, a new fragment must be started.

Okazaki fragments in DNA replication

Okazaki fragments are the short DNA segments produced on the lagging strand. They're roughly 1,000–2,000 nucleotides long in bacteria and 100–200 nucleotides in eukaryotes.

Why do they form? Two constraints collide:

1. DNA strands are antiparallel, so the lagging strand template runs in the "wrong" direction relative to fork movement.
2. DNA polymerase only synthesizes 5'→3'.

The lagging strand gets around this by building DNA in short bursts, each requiring its own start:

1. RNA primase lays down a short RNA primer on the lagging strand template.
2. DNA polymerase III extends from that primer, synthesizing a fragment of DNA in the 5'→3' direction.
3. When the polymerase reaches the RNA primer of the previous fragment, it stops.
4. DNA polymerase I removes the RNA primers and replaces them with DNA.
5. DNA ligase seals the remaining nicks (phosphodiester bond gaps) between adjacent fragments, producing a continuous strand.

Enzymes and Steps in DNA Replication

Key steps of bacterial DNA replication

1. Initiation: DNA helicase binds at the origin of replication and unwinds the double helix, separating the two strands. Single-stranded binding proteins (SSBs) coat the exposed single strands to prevent them from snapping back together (reannealing) or being degraded. Topoisomerase (specifically DNA gyrase in bacteria) relieves the torsional strain ahead of the replication fork that would otherwise cause the DNA to supercoil.

2. Primer synthesis: RNA primase synthesizes short RNA primers complementary to each template strand. DNA polymerase cannot start a new strand from scratch; it needs a free 3'-OH group to add nucleotides to, which the primer provides.

3. Elongation: DNA polymerase III extends the primers by adding deoxyribonucleotides in the 5'→3' direction. On the leading strand, this happens continuously. On the lagging strand, it produces Okazaki fragments.

4. Primer removal and gap filling: DNA polymerase I removes the RNA primers using its 5'→3' exonuclease activity and fills the resulting gaps with DNA.

5. Ligation: DNA ligase catalyzes the formation of phosphodiester bonds between adjacent fragments, sealing any remaining nicks to produce two complete, continuous daughter molecules.

Bacterial vs. eukaryotic DNA replication

Similarities:

• Both use semiconservative replication
• Both are bidirectional with leading and lagging strands
• Both require the same general categories of enzymes (helicase, primase, polymerases, ligase)

Differences:

The multiple origins in eukaryotes compensate for their much larger genomes. Without them, replicating a human chromosome from a single origin would take far too long.

Rolling circle replication mechanism

Rolling circle replication is an alternative mechanism used by some bacteriophages, bacterial plasmids, and certain other genetic elements. Unlike standard bidirectional replication, it produces long, linear concatemers (multiple linked copies of the genome).

Here's how it works:

1. An endonuclease nicks one strand of a circular, double-stranded DNA molecule.
2. The 5' end of the nicked strand is displaced outward as the intact strand serves as a template for new synthesis.
3. DNA polymerase extends the 3' end at the nick site, continuously synthesizing new DNA around the circle. The displaced strand peels off like thread unrolling from a spool.
4. The displaced single strand can itself serve as a template for complementary strand synthesis.
5. The long concatemer is cleaved into individual genome-length units.

This mechanism is particularly important for:

• Single-stranded DNA bacteriophages (e.g., phage φX174) that need to produce many copies quickly
• F plasmid transfer during bacterial conjugation, where rolling circle replication generates the DNA strand transferred to the recipient cell
• Replication of some mitochondrial DNA

DNA replication fidelity and chromosome ends

Accurate replication depends on multiple layers of error prevention:

• Complementary base pairing (A with T, G with C) provides the first level of accuracy. Correct base pairs form more stable hydrogen bonds, so the polymerase preferentially incorporates the right nucleotide.
• DNA polymerase III has 3'→5' exonuclease (proofreading) activity. If a wrong nucleotide is incorporated, the polymerase can back up, remove it, and try again. This reduces the error rate from roughly 1 in $10^5$ to about 1 in $10^7$ base pairs.
• Mismatch repair systems catch errors that escape proofreading, bringing the final error rate down to approximately 1 in $10^9$–$10^{10}$ per base pair per replication.

Telomeres are repetitive DNA sequences at the ends of linear (eukaryotic) chromosomes. They protect coding DNA from the end-replication problem: because the lagging strand needs an RNA primer to start, a small stretch at each chromosome tip can't be fully replicated. Telomeres act as a disposable buffer so that essential genes aren't lost. Bacteria largely avoid this issue because their chromosomes are circular. The enzyme telomerase can extend telomeres in certain eukaryotic cells, but that topic goes beyond standard microbial genetics.

The replication fork is the Y-shaped structure where the parental double helix is actively being unwound and new strands are being synthesized. Each bidirectional origin produces two replication forks moving in opposite directions.