14.3 Basics of DNA Replication

DNA replication is the process by which cells make exact copies of their genetic material before dividing. Understanding how replication works connects DNA's structure (complementary base pairing, antiparallel strands) to its function: faithfully passing genetic information to daughter cells.

DNA Structure and Replication

Structure of DNA for replication

DNA's double helix isn't just a storage shape. Its specific structural features make accurate copying possible.

• Two antiparallel polynucleotide strands wind around each other, with a sugar-phosphate backbone on the outside and nitrogenous bases projecting inward.
• Bases pair through hydrogen bonds following strict rules: adenine (A) with thymine (T) and guanine (G) with cytosine (C). Each pair always involves one purine and one pyrimidine, keeping the helix width uniform.
• Complementary base pairing is what makes semi-conservative replication possible. Because A always pairs with T and G always pairs with C, each single strand already contains the information needed to rebuild its partner.

The two strands run in opposite directions (one 5'→3', the other 3'→5'). This antiparallel orientation has a major consequence for replication: DNA polymerase can only synthesize new DNA in the 5'→3' direction. That means one strand (the leading strand) can be copied continuously, while the other (the lagging strand) must be copied in short pieces called Okazaki fragments.

Key findings of the Meselson-Stahl experiment

Before this experiment, three models of replication were proposed: conservative, semi-conservative, and dispersive. Meselson and Stahl's 1958 experiment distinguished between them.

1. They grew E. coli in a medium containing the heavy nitrogen isotope $^{15}N$, so all the DNA became "heavy."
2. They transferred the bacteria to a medium with normal $^{14}N$ and allowed them to replicate.
3. After one round of replication, all DNA showed a single band at intermediate density on a cesium chloride density gradient. This ruled out the conservative model (which predicted one heavy band and one light band).
4. After a second round, two bands appeared in equal amounts: one at intermediate density and one at light density. This matched the semi-conservative prediction and ruled out the dispersive model.

The conclusion: each new DNA molecule consists of one original (parental) strand and one newly synthesized strand. Density gradient centrifugation was the technique that made it possible to distinguish heavy, intermediate, and light DNA.

Steps in semi-conservative replication

1. Initiation — Replication begins at specific sequences called origins of replication.

   • Helicase unwinds the double helix, creating a replication bubble with a replication fork at each end.
   • Single-stranded binding proteins (SSBs) coat the exposed single strands to prevent them from snapping back together.
   • Topoisomerase relieves the torsional strain ahead of the replication fork caused by unwinding.

2. Primer synthesis — Primase lays down short RNA primers on each template strand.

   • DNA polymerase cannot start a new strand from scratch; it can only add nucleotides to an existing 3' end. The RNA primer provides that starting point.

3. Elongation — DNA polymerases extend the primers by adding nucleotides in the 5'→3' direction.

   • DNA polymerase III copies the leading strand continuously, following right behind the replication fork.
   • On the lagging strand, DNA polymerase III synthesizes short Okazaki fragments (about 1,000–2,000 nucleotides each), each starting from its own RNA primer.
   • DNA polymerase I then removes the RNA primers and replaces them with DNA nucleotides.

4. Joining and termination — Replication continues until the entire molecule is copied.

   • DNA ligase seals the nicks between Okazaki fragments on the lagging strand, creating one continuous strand.
   • In linear chromosomes (eukaryotes), telomerase adds repetitive sequences to chromosome ends so that genetic information isn't lost with each round of replication.

5. Proofreading and error correction — DNA polymerases have built-in quality control.

   • A 3'→5' exonuclease activity lets the polymerase back up and remove a misincorporated nucleotide immediately.
   • Mismatch repair systems catch errors that slip past proofreading, recognizing and excising incorrectly paired bases after replication is complete.
   • Together, these mechanisms keep the error rate remarkably low (roughly 1 error per $10^9$ nucleotides).

DNA Replication Mechanism

A few core terms tie everything together:

• Semiconservative replication: Each daughter DNA molecule keeps one original parental strand paired with one newly made strand. This is the central principle confirmed by Meselson and Stahl.
• Template strand: The existing strand that DNA polymerase "reads" to determine which nucleotide to add next. The new strand is complementary to the template, not identical to it.
• Complementary base pairing: A pairs with T, G pairs with C. This rule is what ensures each copy is accurate.
• Nucleotides: The building blocks added during replication. Each consists of a deoxyribose sugar, a phosphate group, and one of the four nitrogenous bases (A, T, G, or C). They're added to the 3' end of the growing strand, releasing pyrophosphate and providing energy for the reaction.