What you'll learn
DNA structure and replication explains how genetic information is stored and how it is copied with the extraordinary accuracy that inheritance requires. At CAPE level you are expected to know the molecule's structure in chemical detail, to explain how that structure suits each of its functions, and to describe the experimental evidence — notably the Meselson and Stahl experiment — that established semi-conservative replication rather than the competing alternatives. By the end of this topic you should be able to describe nucleotide and DNA structure precisely, apply base pairing rules and Chargaff's data, explain how structure relates to function, describe replication including the roles of each enzyme and the leading and lagging strands, explain the Meselson and Stahl evidence, and compare DNA with RNA.
Key terms and definitions
Nucleotide — the monomer of nucleic acids, consisting of a pentose sugar, a phosphate group and a nitrogenous base
Phosphodiester bond — the covalent bond joining the phosphate of one nucleotide to the sugar of the next
Complementary base pairing — the rule that adenine pairs with thymine and cytosine with guanine
Purine — a base with a double ring structure: adenine and guanine
Pyrimidine — a base with a single ring structure: thymine, cytosine and uracil
Antiparallel — describing the two DNA strands, which run in opposite directions
Semi-conservative replication — replication in which each new molecule contains one original and one newly synthesised strand
Helicase — the enzyme that unwinds the double helix and breaks hydrogen bonds between bases
DNA polymerase — the enzyme that joins free nucleotides to form a new strand
Leading strand — the new strand synthesised continuously towards the replication fork
Lagging strand — the new strand synthesised discontinuously in fragments
Okazaki fragments — the short sections synthesised on the lagging strand
Ligase — the enzyme joining Okazaki fragments together
Mutation — a change in the base sequence of DNA
Core concepts
Nucleotide structure
A nucleotide has three components joined by condensation reactions: a pentose sugar, which is deoxyribose in DNA and ribose in RNA; a phosphate group attached to carbon 5 of the sugar; and a nitrogenous base attached to carbon 1.
Four bases occur in DNA. Adenine and guanine are purines, with a double ring structure. Thymine and cytosine are pyrimidines, with a single ring. In RNA, uracil replaces thymine.
Nucleotides join by condensation between the phosphate group of one and the hydroxyl group on carbon 3 of the sugar of the next, forming a phosphodiester bond and releasing water. The result is a sugar-phosphate backbone with the bases projecting from it.
The double helix
DNA consists of two polynucleotide strands wound into a double helix, with the sugar-phosphate backbones on the outside and the bases paired in the centre.
The two strands are held together by hydrogen bonds between complementary bases. Adenine pairs with thymine by two hydrogen bonds, and cytosine pairs with guanine by three.
Base pairing is complementary because a purine always pairs with a pyrimidine, keeping the distance between the two backbones constant along the whole molecule. Two purines would be too wide and two pyrimidines too narrow.
The strands are antiparallel, running in opposite directions. One runs in the 5 prime to 3 prime direction and the other 3 prime to 5 prime, where the numbers refer to the carbon atoms of the sugar. This orientation is not merely a detail: it is what makes the leading and lagging strands necessary during replication.
Chargaff's rules follow directly from base pairing: in any sample of DNA, the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine. Consequently the total purines equal the total pyrimidines. Questions frequently supply the percentage of one base and ask for the others, and the whole calculation follows from these rules.
Relating structure to function
DNA must do three things, and each is explained by a specific structural feature.
It must store a very large amount of information. The sequence of bases along the molecule carries this information, and because the molecule is extremely long and the bases can occur in any order, an immense number of sequences is possible.
It must be stable, so that information is not corrupted. The sugar-phosphate backbone is held by strong covalent phosphodiester bonds and protects the bases within the helix. The large number of hydrogen bonds, though individually weak, collectively hold the two strands firmly, and the three hydrogen bonds between cytosine and guanine make regions rich in those bases particularly stable.
It must be copied accurately. Complementary base pairing means each strand carries the information needed to reconstruct the other, so each can act as a template. At the same time, the hydrogen bonds are weak enough to be broken by an enzyme when the strands must separate, which is the balance the molecule strikes between stability and accessibility.
Semi-conservative replication
Three models were once possible: conservative, in which the original molecule remains intact and an entirely new one is made; semi-conservative, in which each new molecule has one old and one new strand; and dispersive, in which new and old material is interspersed throughout both strands.
The accepted model is semi-conservative, and the process occurs as follows.
DNA helicase moves along the molecule, breaking the hydrogen bonds between complementary bases and unwinding the double helix. This separates the two strands and forms a replication fork, exposing the bases.
Each original strand acts as a template. Free DNA nucleotides present in the nucleoplasm align opposite their complementary bases by hydrogen bonding — adenine with thymine, cytosine with guanine.
DNA polymerase catalyses the formation of phosphodiester bonds between adjacent nucleotides, building the new strand.
The result is two DNA molecules, each consisting of one original strand and one newly synthesised strand, and each identical to the parent molecule.
Leading and lagging strands
DNA polymerase can only add nucleotides in the 5 prime to 3 prime direction. Because the two template strands are antiparallel, this creates an asymmetry.
On one template, the new strand can be synthesised continuously in the same direction as the replication fork moves. This is the leading strand.
On the other template, synthesis must proceed away from the fork, so it occurs in short sections as the fork opens further. These are Okazaki fragments, and this is the lagging strand. DNA ligase then joins the fragments together by forming phosphodiester bonds between them.
Questions asking why replication differs on the two strands are answered by these two facts together: polymerase works in one direction only, and the strands are antiparallel.
The Meselson and Stahl experiment
This experiment distinguished the three models and is examined in detail.
Bacteria were grown for many generations in a medium containing the heavy nitrogen isotope, so that all their DNA contained heavy nitrogen in its bases. The DNA was extracted and centrifuged in a density gradient, where it formed a single band low in the tube, corresponding to heavy DNA.
The bacteria were then transferred to a medium containing only the normal light isotope and allowed to divide once. The DNA now formed a single band at an intermediate density.
This result immediately eliminated the conservative model, which would have produced two bands — one heavy and one light — after the first generation.
After a second division in light medium, two bands appeared: one at intermediate density and one light, in equal quantities.
This eliminated the dispersive model, which would have produced a single band of steadily decreasing density rather than two distinct bands.
Only the semi-conservative model predicts both results: after one generation every molecule has one heavy and one light strand, giving intermediate density; after two generations half the molecules are intermediate and half are entirely light.
Mutation and proofreading
Replication is extremely accurate, partly because DNA polymerase proofreads as it works, checking that each added nucleotide is correctly paired and removing mismatches.
Errors that remain are mutations, changes in the base sequence. A substitution replaces one base with another and may or may not change the amino acid coded for, because the genetic code is degenerate. An insertion or deletion adds or removes a base and causes a frameshift, altering every codon downstream, and is therefore usually far more damaging.
Mutation rate is increased by mutagens such as ionising radiation, ultraviolet light and certain chemicals.
Comparing DNA and RNA
DNA has deoxyribose sugar, the bases adenine, thymine, cytosine and guanine, two strands in a double helix, and is very long and stable; its function is the long-term storage of genetic information in the nucleus.
RNA has ribose sugar, uracil in place of thymine, a single strand, and is much shorter and less stable; its function is to carry and interpret the genetic message.
Three types of RNA matter. Messenger RNA carries the code from nucleus to ribosome. Transfer RNA carries specific amino acids to the ribosome and has an anticodon complementary to a codon on the messenger RNA. Ribosomal RNA forms part of the structure of the ribosome.
Worked examples
Example 1: Applying Chargaff's rules (4 marks)
A sample of double-stranded DNA contains 22 per cent adenine. Calculate the percentage of each of the other three bases, showing your reasoning.
By complementary base pairing, adenine pairs only with thymine, so the amount of thymine equals the amount of adenine. Thymine is therefore 22 per cent.
Adenine and thymine together account for 22 + 22 = 44 per cent of the bases. The remaining 56 per cent must be cytosine and guanine.
Cytosine pairs only with guanine, so these two are present in equal amounts. Each is therefore 56 ÷ 2 = 28 per cent.
The answer is thymine 22 per cent, cytosine 28 per cent and guanine 28 per cent.
Example 2: Explaining the Meselson and Stahl result (5 marks)
Explain how the appearance of a single band of intermediate density after one generation, and two bands after two generations, supports semi-conservative replication.
After growth in heavy nitrogen, all DNA strands contained the heavy isotope. After one division in light medium, semi-conservative replication predicts that every molecule contains one original heavy strand and one newly synthesised light strand, giving every molecule the same intermediate density and therefore a single intermediate band, which is what was observed.
The conservative model predicts that the original heavy molecule would remain intact while an entirely new light molecule was made, giving two bands after one generation. Since only one band appeared, this model is eliminated.
After a second division, each intermediate molecule separates into one heavy and one light strand, each templating a new light strand. This gives half the molecules as intermediate and half as entirely light, producing two bands in equal quantities, as observed.
The dispersive model predicts that old and new material would be mixed throughout every strand, so all molecules would have the same density at each generation, giving a single band of progressively decreasing density rather than two distinct bands. This result therefore eliminates it too, leaving only the semi-conservative model.
Example 3: Explaining the lagging strand (4 marks)
Explain why one new DNA strand is synthesised continuously and the other in fragments.
DNA polymerase can only add nucleotides to the 3 prime end of a growing strand, so it can only synthesise in the 5 prime to 3 prime direction.
The two template strands are antiparallel, running in opposite directions. On one template, the 5 prime to 3 prime direction of synthesis is the same as the direction in which the replication fork is opening, so the new strand can be made continuously as the fork advances. This is the leading strand.
On the other template, the 5 prime to 3 prime direction of synthesis points away from the fork. Synthesis can therefore only begin after a length of template has been exposed, and must proceed backwards, producing short Okazaki fragments. DNA ligase then joins these fragments by forming phosphodiester bonds between them.
Common mistakes and how to avoid them
The most frequent error is stating that the two strands are joined by hydrogen bonds between the sugar-phosphate backbones. The hydrogen bonds are between the complementary bases; the backbones are held by covalent phosphodiester bonds within each strand.
Students often give the wrong number of hydrogen bonds: adenine and thymine have two, cytosine and guanine three.
Another common slip is confusing the roles of helicase and polymerase. Helicase unwinds and separates; polymerase joins nucleotides.
Many candidates describe replication as producing two entirely new molecules. Each new molecule contains one original strand, which is precisely what semi-conservative means.
In Meselson and Stahl questions, answers frequently describe the results without saying which model each result eliminates. The elimination is where the marks are.
Finally, candidates often state that DNA polymerase works in both directions. It works only 5 prime to 3 prime, which is the entire reason for the lagging strand.
Exam technique for "DNA structure and replication"
When asked to relate structure to function, pair each structural feature with the function it enables: base sequence with information storage, phosphodiester bonds with stability, complementary pairing with accurate copying.
For base percentage calculations, state Chargaff's rules explicitly before calculating. The reasoning carries marks even when the arithmetic is trivial.
Name every enzyme in a replication answer and give its specific action. Helicase, DNA polymerase and ligase are three separate marks.
For the Meselson and Stahl experiment, structure the answer by model: state what each model predicts and what the observed result shows about it.
Use the terms 5 prime and 3 prime when explaining leading and lagging strands. Answers that avoid the directional terms cannot fully explain the asymmetry.
Quick revision summary
A nucleotide is a pentose sugar, a phosphate and a nitrogenous base, joined into strands by phosphodiester bonds between the phosphate of one and carbon 3 of the next. DNA is a double helix with sugar-phosphate backbones outside and paired bases inside, adenine bonding to thymine with two hydrogen bonds and cytosine to guanine with three, purines always pairing with pyrimidines so the helix width stays constant. The strands are antiparallel. Chargaff's rules state that adenine equals thymine and cytosine equals guanine. The base sequence stores information, phosphodiester bonds and numerous hydrogen bonds give stability, and complementary pairing allows each strand to template the other. Replication is semi-conservative: helicase breaks hydrogen bonds and unwinds, free nucleotides align with complementary bases, and DNA polymerase forms phosphodiester bonds, working only 5 prime to 3 prime so that one strand is continuous and the other is made as Okazaki fragments joined by ligase. Meselson and Stahl showed one intermediate band after one generation, eliminating the conservative model, and two bands after two, eliminating the dispersive model. RNA differs in having ribose, uracil, a single strand and a shorter, less stable structure.