Describe the structure of DNA, the process of semi-conservative replication and the role of key enzymes including helicase, DNA polymerase, primase and ligase

What this dot point is asking

QCAA wants you to describe the molecular structure of DNA, explain why replication is described as semi-conservative, and name the enzymes that do each job at the replication fork. Diagrams of the double helix and the replication fork are common stimulus material.

The answer

DNA is the molecule that stores hereditary information in all cellular life. Its structure is elegantly suited to two jobs: storing information stably, and being copied accurately every time a cell divides.

The structure of DNA

DNA is a polymer of nucleotides. Each nucleotide has three parts:

• A deoxyribose sugar (five-carbon sugar with a hydroxyl group on carbon 3 and a hydrogen on carbon 2).
• A phosphate group attached to carbon 5 of the sugar.
• A nitrogenous base attached to carbon 1.

There are four bases, in two chemical classes:

• Purines (double-ring): adenine (A) and guanine (G).
• Pyrimidines (single-ring): thymine (T) and cytosine (C).

Nucleotides join into a single strand through phosphodiester bonds between the phosphate of one nucleotide and the 3 prime hydroxyl of the next. This gives every strand a direction, called 5 prime to 3 prime.

Two strands then pair to form the double helix, held together by hydrogen bonds between complementary bases:

• A pairs with T using two hydrogen bonds.
• C pairs with G using three hydrogen bonds.

The two strands are antiparallel: one runs 5 prime to 3 prime, the other 3 prime to 5 prime. The whole structure twists into a right-handed helix, about 10 base pairs per turn, with the sugar to phosphate backbone on the outside and the bases stacked on the inside.

Why the structure works for storage and copying.

• Hydrogen bonds are strong enough collectively to hold the helix together at body temperature, but individually weak enough to be broken by enzymes when the molecule is read or copied.
• Complementary base pairing means each strand carries the full information needed to rebuild the other.
• The sugar to phosphate backbone is chemically uniform, so any sequence of bases can be stored without changing the physical shape of the helix.

Semi-conservative replication

When a cell divides, every DNA molecule must be copied. The copy mechanism is semi-conservative: each daughter double helix contains one parental (original) strand and one newly synthesised strand.

Two alternative models existed historically:

• Conservative. The parental helix stays intact and the daughter helix is entirely new.
• Dispersive. Strands are broken into fragments that mix old and new DNA.

The famous Meselson and Stahl (1958) experiment used the heavy nitrogen isotope 15N. E. coli grown in 15N medium had heavy DNA; transferred to 14N medium and allowed to replicate, the DNA was sampled at each generation and separated by density gradient centrifugation:

• After one generation: a single band of intermediate density. (Rules out conservative.)
• After two generations: two bands, one intermediate and one light. (Rules out dispersive.)

Only the semi-conservative model predicted this exact pattern.

The replication fork

Replication begins at a specific sequence called the origin of replication. Several enzymes act at the replication fork where the helix opens.

Helicase

Breaks hydrogen bonds between paired bases and unwinds the helix. Single-strand binding proteins keep the separated strands from re-annealing. Topoisomerase relieves the supercoiling tension ahead of the fork.

Primase

Synthesises a short RNA primer (about 5 to 10 ribonucleotides) complementary to the template. The primer provides a free 3 prime hydroxyl group, which is what DNA polymerase needs to extend from.

DNA polymerase III

The main copying enzyme in prokaryotes (eukaryotes use a family of polymerases). It adds DNA nucleotides to the 3 prime end of the growing strand, reading the template 3 prime to 5 prime and synthesising the new strand 5 prime to 3 prime. Each new nucleotide arrives as a deoxynucleoside triphosphate (dATP, dTTP, dCTP, dGTP); two phosphates are cleaved off, releasing energy for the bond. DNA polymerase also proofreads, removing mismatched bases as it goes.

Leading and lagging strands

Because the two template strands are antiparallel, only one of them can be copied continuously toward the fork. This is the leading strand. The other, the lagging strand, is copied in short pieces called Okazaki fragments, each starting from a new primer. The lagging strand needs many primers.

DNA polymerase I

Removes the RNA primers and fills the gaps with DNA.

Ligase

Seals the nicks between Okazaki fragments by forming the final phosphodiester bond, producing one continuous strand.

Accuracy

DNA polymerase has a built-in proofreading function: it checks each newly added nucleotide and excises mistakes (3 prime to 5 prime exonuclease activity). Combined with mismatch repair after replication, the overall error rate is about one mistake in a billion bases.

Examples in context

Example 1. Mt Isa lead-tolerant bacterial DNA repair. Cupriavidus metallidurans from Mt Isa tailings replicates its 6 megabase chromosome roughly every 50 minutes. Helicase unwinds the double helix at the replication fork by breaking hydrogen bonds between complementary bases (A=T two bonds, G=C three bonds). DNA polymerase III synthesises the leading strand continuously and the lagging strand as Okazaki fragments, each primed by primase. Ligase seals the nicks. Lead exposure raises DNA damage rates; the bacterium upregulates recA and proofreading exonucleases to maintain fidelity. The Mt Isa strains thrive at lead concentrations 1000 times above what kills laboratory E. coli, showing that the same replication machinery operates under extreme selective pressure.

Example 2. Meselson-Stahl revisited in Brisbane teaching labs. University of Queensland teaching laboratories demonstrate the semi-conservative model by growing E. coli in nitrogen-15 ($^{15}$N) medium for many generations, then switching to nitrogen-14 ($^{14}$N) and sampling after 0, 1 and 2 generations. After one round of replication, DNA shows a single intermediate-density band on caesium chloride gradients; after two rounds, two bands appear (intermediate plus light). The 1958 result remains the classroom benchmark for semi-conservative replication and lets students see that each daughter molecule contains one parental and one new strand, consistent with the action of helicase, polymerase, primase and ligase at the fork.

Try this

Q1. Describe the role of helicase, primase, DNA polymerase and ligase in semi-conservative replication. [4 marks]

• Cue. Helicase unwinds; primase makes RNA primer; polymerase extends; ligase seals Okazaki fragments.

Q2. A DNA strand has the sequence 5'-AGCTTAGC-3'. Write the complementary strand and the mRNA that would result if the original strand were transcribed. [3 marks]

• Cue. Complement DNA 3'-TCGAATCG-5'; mRNA 5'-AGCUUAGC-3' (if other strand is template, otherwise rewrite).

Q3. Refer to leading and lagging strand synthesis. (a) Explain why one strand is synthesised continuously. (b) Identify the role of Okazaki fragments. (c) Predict the effect of a ligase mutation. [2+2+2 marks]

• Cue. (a) Polymerase only works 5' to 3'. (b) Lagging strand short pieces. (c) Nicks accumulate; chromosome fragments.