12 min readPathway sheet1-mark MCQ, a 3-mark question on Meselson and Stahl's experiment, and a 5-mark answer with a labelled replication fork; also a NEET favourite

DNA replication: semiconservative copying

DNA copies itself semiconservatively: each new molecule keeps one old strand. DNA polymerase works only 5′→3′, so one strand is made in short pieces joined by ligase.

Semiconservative replicationEach daughter DNA has one parental (old) strand and one newly made strand

Main enzymeDNA-dependent DNA polymerase; adds nucleotides only in the 5′→3′ direction

Substrates and energyDeoxyribonucleoside triphosphates act as both substrates and energy source

E. coli speed4.6 × 10⁶ bp copied in about 18 minutes; about 2000 bp per second

Meselson and Stahl's experiment (1958)

1
E. coli was grown for many generations in ¹⁵NH₄Cl, so all its DNA had heavy nitrogen (¹⁵N). ¹⁵N is a heavy isotope, not a radioactive one.
2
The cells were moved to a medium with normal ¹⁴NH₄Cl. Samples were taken as the cells divided, about every 20 minutes.
3
DNA was extracted and spun in a caesium chloride (CsCl) density gradient. Heavy, hybrid and light DNA settle as bands at different heights.
4★
After one generation (20 minutes): a single band of hybrid (intermediate) density, ¹⁵N/¹⁴N.
5
After two generations (40 minutes): equal amounts of hybrid and light DNA.
6
Only semiconservative replication explains this. In the same year, Taylor and colleagues showed the same in Vicia faba (faba bean) chromosomes, using radioactive thymidine.

★ key step

Predicting the bands after three generations

Problem

In Meselson and Stahl's experiment, what fraction of DNA molecules will be (a) hybrid and (b) light after three generations in ¹⁴N medium?

Working

Start: 1 heavy molecule, both strands ¹⁵N.
Generation 1: 2 molecules, both hybrid.
Generation 2: 4 molecules: 2 hybrid and 2 light.
Generation 3: 8 molecules. The two old ¹⁵N strands are still in 2 hybrid molecules; the other 6 are light.
Hybrid = 2/8 = 1/4; light = 6/8 = 3/4.

Answer

(a) 1/4 hybrid (25%), (b) 3/4 light (75%). No fully heavy DNA is left after the first generation.

The machinery and the replication fork

1
Replication begins at a fixed region called the origin of replication (ori).
2
The long helix cannot be unwound all at once because that needs too much energy. So the strands separate at a small opening, the replication fork.
3
DNA-dependent DNA polymerase uses each old strand as a template and adds deoxyribonucleoside triphosphates, which also supply the energy.
4★
The polymerase works only 5′→3′. On the template with 3′→5′ polarity, the new strand grows continuously (leading strand).
5
On the template with 5′→3′ polarity, the new strand is made discontinuously in short pieces (Okazaki fragments); this is the lagging strand.
6
DNA ligase joins these short pieces.
7
In eukaryotes, replication happens in the S phase of the cell cycle. If the cell then fails to divide, the result is polyploidy.

★ key step

Leading strand vs lagging strand

Leading strandMade on the template with 3′→5′ polarity. Grows continuously towards the replication fork.

Lagging strandMade on the template with 5′→3′ polarity. Made discontinuously as short pieces away from the fork, which DNA ligase joins.

Why the textbook says about 2000 bp per second

Problem

E. coli has 4.6 × 10⁶ bp and copies them in about 18 minutes, starting from one origin. Assume two forks move away from the origin in opposite directions. Find the rate at each fork.

Working

Time = 18 × 60 = 1080 s.
The two forks share the work, so each copies 4.6 × 10⁶ ÷ 2 = 2.3 × 10⁶ bp.
Rate at each fork = 2.3 × 10⁶ ÷ 1080 ≈ 2130 bp per second.
Rounded, this is about 2000 bp per second.

Answer

About 2 × 10³ bp per second at each fork (2130, rounded to one significant figure).

How to draw a replication fork

Draw the parent double helix on one side; label 5′ and 3′ on each strand, in opposite directions.
Open the two strands into a Y shape; label the junction replication fork.
On the template running 3′→5′, draw one long new strand with an arrow towards the fork; label leading strand (continuous).
On the other template, draw short new pieces with arrows pointing away from the fork; label lagging strand (discontinuous) and Okazaki fragments.
Label DNA polymerase on the new strands and DNA ligase at a join between two pieces.
Write 5′ and 3′ at the ends of every new strand too.

In real life

Some anti-cancer drugs block DNA replication, so fast-dividing cancer cells are hit hardest. That is also why the fast-dividing cells of hair roots and the gut lining are affected during treatment.

Exam angle

3 marks: “Describe Meselson and Stahl's experiment. What would you see in the CsCl gradient after 40 minutes?” E. coli grown in ¹⁵NH₄Cl, moved to ¹⁴NH₄Cl; DNA spun in a CsCl density gradient; after 20 minutes all hybrid; after 40 minutes equal hybrid and light bands. This proves semiconservative replication.

2 marks: “Why is replication continuous on one template strand and discontinuous on the other?” DNA polymerase adds nucleotides only 5′→3′. On the 3′→5′ template it can follow the fork continuously; on the 5′→3′ template it must work in short pieces, later joined by DNA ligase.

5 marks: “Explain the process of DNA replication with a labelled diagram of a replication fork.” Semiconservative; origin of replication; replication fork; DNA-dependent DNA polymerase, dNTPs as substrate and energy; 5′→3′ only; continuous and discontinuous strands; ligase; S phase; labelled fork.

Remember it

Polymerase only goes 5′ to 3′: one strand runs smoothly, the other goes in bits, and ligase glues the bits.