7.2 DNA Replication Process

DNA replication is the process cells use to make an exact copy of their DNA before cell division. Without it, daughter cells wouldn't receive the complete set of genetic instructions they need to function. Understanding how replication works also helps explain where mutations come from and why they're relatively rare.

DNA Replication Enzymes

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Essential Enzymes for DNA Replication

Each enzyme in replication has a specific job. Think of it like an assembly line where every worker handles one task, and the whole process falls apart if any single one is missing.

• DNA helicase unwinds the double helix by breaking the hydrogen bonds between complementary base pairs, separating the two strands so they can each be copied.
• Primase synthesizes short RNA primers (about 8–12 nucleotides long) that give DNA polymerase a starting point. DNA polymerase can't start a new strand from scratch; it can only add nucleotides to an existing strand, so these primers are essential.
• DNA polymerase reads the template strand and adds complementary nucleotides to the growing chain, always building in the 5' to 3' direction. This directionality is a big deal and directly causes the leading/lagging strand difference covered below.
• DNA ligase seals the gaps between Okazaki fragments on the lagging strand by forming phosphodiester bonds between the 3' end of one fragment and the 5' end of the next, creating one continuous strand.

Proofreading and Error Correction

DNA polymerase doesn't just build new strands; it also proofreads as it goes. If it detects a mismatched nucleotide, it can reverse direction, remove the incorrect base, and replace it with the right one. This is called 3' to 5' exonuclease activity.

Even after proofreading, some errors slip through. A second layer of defense called mismatch repair catches these. In E. coli, enzymes like MutS and MutL scan newly made DNA, recognize mismatches, and fix them. Together, proofreading and mismatch repair bring the final error rate down to roughly one mistake per $10^9$ (one billion) base pairs copied.

Replication Process

Semiconservative Replication and the Replication Fork

DNA replication is semiconservative, meaning each of the two resulting DNA molecules contains one original ("parent") strand and one newly synthesized strand. This was confirmed by the Meselson-Stahl experiment in 1958, which used heavy nitrogen ($^{15}N$) to distinguish old strands from new ones.

Here's how the process unfolds:

1. Replication begins at a specific DNA sequence called the origin of replication. Eukaryotic chromosomes have many origins so that their large genomes can be copied quickly.
2. Helicase binds at the origin and unwinds the double helix in both directions, creating two Y-shaped replication forks that move away from each other.
3. Single-strand binding proteins (SSBPs) stabilize the exposed single strands and prevent them from snapping back together.
4. Each exposed strand now serves as a template for building a new complementary strand.

Leading and Lagging Strands

Because DNA polymerase can only synthesize in the 5' to 3' direction, the two template strands at each replication fork are copied differently.

• Leading strand: The template here runs 3' to 5' toward the fork, so DNA polymerase can follow the fork and synthesize one long, continuous strand. It needs only a single RNA primer to get started.
• Lagging strand: The template runs 5' to 3' toward the fork, which means DNA polymerase has to work away from the fork. It synthesizes short pieces called Okazaki fragments (about 1,000–2,000 nucleotides in eukaryotes, 100–200 in prokaryotes). Each fragment requires its own RNA primer from primase.

After Okazaki fragments are made, the RNA primers are removed and replaced with DNA (by DNA polymerase), and then DNA ligase seals the fragments together into a continuous strand.