28.3 Replication of DNA

DNA replication is the process cells use to make an exact copy of their DNA before dividing. Understanding the chemistry behind each step is essential for organic chemistry, since the mechanism depends on enzyme specificity, hydrogen bonding between base pairs, and the directionality of phosphodiester bond formation.

DNA Structure and Replication Components

Before diving into the replication mechanism, you need a solid handle on DNA's structure.

Each nucleotide has three parts: a deoxyribose sugar, a phosphate group, and a nitrogenous base. Adjacent nucleotides are linked by phosphodiester bonds between the 3'-hydroxyl of one sugar and the 5'-phosphate of the next, forming the sugar-phosphate backbone.

The two strands of DNA are antiparallel, meaning one runs 5'→3' while the other runs 3'→5'. They're held together by hydrogen bonds between complementary bases:

• Adenine (A) pairs with Thymine (T) via 2 hydrogen bonds
• Guanine (G) pairs with Cytosine (C) via 3 hydrogen bonds

This antiparallel arrangement is the root cause of why the two strands are replicated differently, as you'll see below.

DNA Replication

Semiconservative Nature of Replication

Each new DNA double helix contains one original (parental) strand and one newly synthesized (daughter) strand. During replication, the parental strands separate, and each serves as a template for building a new complementary strand. The result is two identical double helices, each half-old and half-new.

This was demonstrated by the Meselson-Stahl experiment (1958). They grew bacteria in medium containing heavy nitrogen ($^{15}N$), then switched to light nitrogen ($^{14}N$). After one round of replication, all DNA had an intermediate density, ruling out both conservative and dispersive models. After two rounds, half the DNA was intermediate and half was light, exactly what semiconservative replication predicts.

Process of DNA Replication

Here's the step-by-step mechanism:

1. Initiation at origins of replication. Replication begins at specific DNA sequences called origins of replication, where the double helix first opens up.

2. Helicase unwinds the double helix. Helicase breaks the hydrogen bonds between complementary base pairs, separating the two strands and creating a replication fork (the Y-shaped region where unwinding is actively occurring).

3. Stabilization and tension relief.

   • Single-stranded binding proteins (SSBs) coat the exposed single strands to prevent them from re-annealing or being degraded by nucleases.
   • Topoisomerase works ahead of the replication fork to relieve the torsional strain (supercoiling) that builds up as the helix unwinds.

4. Primer synthesis. DNA primase synthesizes short RNA primers (roughly 10 nucleotides) complementary to the template strand. These primers are necessary because DNA polymerase cannot start a new strand from scratch; it can only add nucleotides to an existing 3'-OH group.

5. Elongation by DNA polymerase III. This is the main replication enzyme. It reads the template strand 3'→5' and synthesizes the new strand 5'→3', adding deoxyribonucleotides that are complementary to the template. Each incoming nucleotide forms a phosphodiester bond with the 3'-OH of the growing chain.

6. Primer removal by DNA polymerase I. Once synthesis is underway, DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides, using its 5'→3' exonuclease activity.

7. Ligation. DNA ligase catalyzes the formation of a phosphodiester bond to seal the remaining nicks (single-strand breaks) in the backbone, producing a continuous strand.

Leading vs. Lagging Strand Synthesis

Because DNA polymerase can only synthesize in the 5'→3' direction, and the two template strands run in opposite directions, the two new strands are built by different strategies.

Leading strand:

• Synthesized continuously in the 5'→3' direction, which happens to be the same direction the replication fork is moving.
• Only one RNA primer is needed. DNA polymerase III extends it smoothly as the fork opens.

Lagging strand:

• Synthesized discontinuously in short segments called Okazaki fragments, because the 5'→3' synthesis direction is opposite to the direction of fork movement.
• The process works like this:
  1. Primase lays down an RNA primer on the lagging strand template.
  2. DNA polymerase III extends the primer 5'→3', away from the fork, until it reaches the previous fragment.
  3. This cycle repeats, producing multiple Okazaki fragments (about 100–200 nucleotides in eukaryotes, 1,000–2,000 in prokaryotes).
  4. DNA polymerase I removes each RNA primer and fills the gap with DNA.
  5. DNA ligase seals the nicks between adjacent fragments, creating a continuous strand.

Replication is also bidirectional: two replication forks move in opposite directions from each origin, so both sides of the origin are replicated simultaneously. At each fork, one strand is the leading strand and the other is the lagging strand.