Semiconservative DNA Replication

Semiconservative DNA replication is the process by which a cell copies its DNA before division, producing two identical molecules, each made of one original (old) strand and one newly built strand. In brief: helicase unwinds the double helix, DNA polymerase builds new strands by adding nucleotides complementary to each template strand, and ligase joins fragments. The copying is highly accurate, with about one error per billion bases after proofreading and repair.

Replication ensures that every daughter cell receives a complete and accurate copy of the genetic information. This guide describes the experiment that showed the semiconservative model, the enzymes and steps of replication, the leading and lagging strands, how errors are controlled, works through a base-pairing and a Meselson-Stahl example and corrects common mistakes.

Why is replication called semiconservative?

Three models were proposed for how DNA could be copied. In the conservative model, the original double helix stays intact and a completely new copy is made. In the dispersive model, the old and new DNA are mixed in each strand. In the semiconservative model, the double helix separates and each strand serves as a template for a new complementary strand. The result is two molecules, each with one old and one new strand. Experiments have shown that the semiconservative model is correct.

The model follows from complementary base pairing: because A always pairs with T and C always pairs with G, each old strand fully determines the sequence of its partner.

What are the steps of DNA replication?

1. Initiation: replication starts at specific sequences called origins of replication. Bacteria usually have one origin on their circular chromosome, and eukaryotes have thousands along each chromosome. This makes copying faster. A human cell copies about 6 billion base pairs in around 8 hours of S phase.
2. Unwinding: the enzyme helicase breaks the hydrogen bonds between bases and separates the strands, creating a replication fork. Single-strand binding proteins keep the strands apart. Topoisomerase relieves the tension that builds up ahead of the fork.
3. Priming: the enzyme primase makes a short RNA primer that gives DNA polymerase a starting point, because DNA polymerase can only add nucleotides to an existing 3′ end.
4. Elongation: DNA polymerase adds free nucleotides to the 3′ end of the new strand, matching each template base (A with T, C with G). The energy comes from the nucleotides themselves, which are supplied as triphosphates.
5. Joining and termination: RNA primers are removed and replaced with DNA, and the enzyme DNA ligase seals the gaps between fragments. In eukaryotes, special ends of chromosomes called telomeres are maintained by an enzyme called telomerase.

Which enzymes are involved?

Replication requires the coordinated action of several enzymes. The key ones are listed in the table below.

What are the leading and lagging strands?

DNA polymerase can add nucleotides only to the 3′ end, so a new strand is always built in the 5′ to 3′ direction. Because the two template strands are antiparallel, the two new strands are made in different ways at the replication fork.

• The leading strand is made continuously, in the same direction as the opening of the fork, with one primer.
• The lagging strand is made discontinuously in short pieces called Okazaki fragments, each starting from its own primer, in the direction away from the fork. The fragments are about 100 to 200 nucleotides long in eukaryotes (about 1 000 to 2 000 in bacteria). They are later joined by ligase.

How accurate is replication?

DNA polymerase inserts the wrong nucleotide about once every 10 000 to 100 000 bases. It also has a proofreading function: it can detect a mismatch, move back and remove the wrong nucleotide, which reduces the error rate to about one in 10 million. Mismatch repair proteins then scan the new strand and correct most of the remaining errors, giving a final rate of roughly one error per billion nucleotides copied. The rare errors that remain are mutations, the source of new alleles. A mutation in a gene for repair proteins can increase the chance of cancer.

Replication enzymes at a glance

Worked example: tracing the strands

A short piece of DNA has the following strands (written with the top strand 5′ to 3′ and the bottom strand 3′ to 5′, aligned):

Top: 5′-ATGCGT-3′
Bottom: 3′-TACGCA-5′

1. Separate the strands: helicase separates the top strand and the bottom strand.
2. Copy the top strand: each base in the top strand is paired with its complement. The new strand is 3′-TACGCA-5′ (A–T, T–A, G–C, C–G, G–C, T–A).
3. Copy the bottom strand: the new strand is 5′-ATGCGT-3′ (T–A, A–T, C–G, G–C, C–G, A–T).
4. Result: two molecules, each identical to the original. Molecule 1 consists of the old top strand (5′-ATGCGT-3′) and the new bottom strand. Molecule 2 consists of the old bottom strand (3′-TACGCA-5′) and the new top strand.

Each new molecule contains one old strand and one new strand, which is the definition of semiconservative replication. Counting the nucleotides that need to be added: for a 6-base-pair fragment, 12 nucleotides are added in total (6 for each template strand). For a human cell with a diploid genome of about 6.4 billion base pairs, the S phase adds about 12.8 billion nucleotides, which doubles the amount of DNA in the nucleus, as described in the cell cycle article.

What did the Meselson-Stahl experiment show?

In 1958, Matthew Meselson and Franklin Stahl grew E. coli for many generations in a medium with a heavy isotope of nitrogen (¹⁵N), so all the DNA was heavy. They moved the bacteria to a medium with normal nitrogen (¹⁴N) and took samples after each generation. DNA was separated by density in a centrifuge.

1. Generation 0: all DNA was heavy (¹⁵N/¹⁵N), forming a band low in the tube.
2. Generation 1: all DNA formed a single band at an intermediate density (¹⁵N/¹⁴N). This ruled out the conservative model, which predicts one heavy and one light band.
3. Generation 2: two bands: one intermediate (¹⁵N/¹⁴N) and one light (¹⁴N/¹⁴N), in equal amounts. This ruled out the dispersive model, which predicts a single band of lower density, and supported the semiconservative model.

The quantity of each type of DNA in later generations can be predicted. After n generations, the fraction of DNA molecules that contain one heavy strand is 2 ÷ 2ⁿ. After 3 generations: 2 ÷ 8 = 25 percent are hybrid, and 75 percent are light. The experiment is one of the most elegant in the history of biology and was called “the most beautiful experiment in biology”.

Where is replication used in technology?

The polymerase chain reaction (PCR) copies DNA in the laboratory using the principles of replication, repeated cycles of heating and cooling and a heat-stable DNA polymerase. PCR is used in medical diagnosis, forensic analysis and research. DNA sequencing methods also use polymerase to build complementary strands. Antiviral and anticancer drugs often target replication enzymes, as in the case of some drugs that stop DNA polymerase of viruses.

Which mistakes are common?

Mistake 1: Saying that one new double helix is made and the old one is kept

This is the conservative model, which is wrong. In semiconservative replication each new helix has one old strand.

Mistake 2: Saying that DNA polymerase starts replication

Helicase opens the helix and primase makes the primer first. DNA polymerase extends the primer.

Mistake 3: Thinking that both strands are made continuously

Only the leading strand is continuous. The lagging strand is made in fragments.

Mistake 4: Mixing up the 5′ and 3′ ends

New nucleotides are always added to the 3′ end, so strands grow 5′ to 3′.