Structure of nucleic acids and replication of DNA

Nucleic acids (DNA and RNA) are polymers of nucleotides. Each nucleotide has three components joined by condensation reactions:

1. Pentose sugar — a five-carbon sugar. DNA contains deoxyribose; RNA contains ribose (which has an extra –OH on the 2' carbon).
2. Phosphate group — attached to the 5' carbon of the pentose. It is acidic and gives DNA its overall negative charge.
3. Nitrogenous base — attached to the 1' carbon of the pentose. There are five bases in total: adenine (A), thymine (T), cytosine (C), guanine (G) in DNA, plus uracil (U) which replaces T in RNA.

Bases fall into two structural classes:

• Purines — adenine (A) and guanine (G) — have a double-ring structure.
• Pyrimidines — cytosine (C), thymine (T) and uracil (U) — have a single-ring structure.

Nucleotides are joined together by phosphodiester bonds between the 3'-OH of one sugar and the 5'-phosphate of the next. These bonds are formed by condensation (with loss of water) and broken by hydrolysis. The resulting sugar-phosphate backbone runs along the outside of the polynucleotide strand, with the bases projecting inwards.

Each polynucleotide strand has a free 5' phosphate end and a free 3' hydroxyl end, giving it directionality.

DNA exists as a double helix — two polynucleotide strands wound around a common axis. The structure was deduced in 1953 by Watson and Crick, using X-ray diffraction data from Rosalind Franklin and Maurice Wilkins.

Key structural features:

• The two strands are antiparallel: one runs 5'→3' and the other runs 3'→5'. This orientation is essential for correct base pairing.
• The sugar-phosphate backbones are on the outside of the helix; the bases project inwards and pair across the centre.
• The strands are held together by hydrogen bonds between complementary bases:
  • A pairs with T by 2 hydrogen bonds.
  • G pairs with C by 3 hydrogen bonds.
• A purine always pairs with a pyrimidine, which keeps the helix at a constant width of about 2 nm.
• The helix completes one full turn every ~10 base pairs (~3.4 nm of length).
• The structure has a major groove and a minor groove along its surface — sites where regulatory proteins can read the base sequence without unwinding the DNA.

The hydrogen bonds are individually weak, but there are millions of them along a typical DNA molecule, making the helix very stable. The bonds can also be broken in a controlled way by enzymes (helicase, RNA polymerase) when the DNA needs to be unwound for replication or transcription.

The base sequence along one strand carries the genetic information of the cell. Because the strands are complementary, the sequence of one strand uniquely determines the sequence of the other — this is the structural basis of DNA replication and transcription.

Both DNA and RNA are polynucleotides but differ structurally in four important ways:

Feature	DNA	RNA
Pentose sugar	Deoxyribose (no 2'-OH)	Ribose (has 2'-OH)
Bases	A, T, C, G	A, U, C, G
Number of strands	Double (double helix)	Single
Length	Very long (millions of bp)	Much shorter (hundreds to a few thousand)
Stability	Very stable	Less stable (2'-OH makes ribose more reactive)
Function	Permanent store of genetic information	Temporary working copies — mRNA, tRNA, rRNA

There are three main types of RNA, each with a specific role in protein synthesis (covered in the next subtopic):

• mRNA (messenger RNA) — carries the genetic code from DNA to the ribosome.
• tRNA (transfer RNA) — folded clover-leaf shape; carries a specific amino acid to the ribosome and matches its anticodon to the mRNA codon.
• rRNA (ribosomal RNA) — together with proteins forms the ribosome; rRNA also catalyses peptide bond formation.

Because RNA is less stable, it is suited to its role as a working / disposable copy: mRNA is degraded shortly after use so that the cell can quickly adjust which genes are being expressed.

DNA replication takes place during the S phase of the cell cycle, before mitosis or meiosis. The process is described as semi-conservative because each of the two daughter DNA molecules contains one parental strand and one newly synthesised strand.

The steps of replication are:

1. Unwinding. The enzyme DNA helicase moves along the parental double helix, breaking the hydrogen bonds between complementary bases. The two strands separate at the replication fork, each becoming a template.
2. Free nucleotide alignment. Free DNA nucleotides (containing all four bases) are present in the nucleoplasm. They line up against the exposed bases of each template strand by complementary base pairing — A with T, G with C.
3. Polymerisation. The enzyme DNA polymerase catalyses the formation of phosphodiester bonds between the 3'-OH of the growing chain and the 5'-phosphate of the next nucleotide. DNA polymerase can only add nucleotides in the 5'→3' direction.
4. Leading and lagging strands. Because the two templates are antiparallel:
   • The leading strand template (the 3'→5' template) is read continuously and the new strand grows in step with the replication fork.
   • The lagging strand template (the 5'→3' template) cannot be read continuously, because synthesis must still proceed 5'→3'. The new strand is made in short pieces called Okazaki fragments that point away from the fork.
5. Joining fragments. The enzyme DNA ligase seals the gaps between Okazaki fragments by forming the final phosphodiester bonds.
6. Result. Two daughter DNA molecules are produced, each identical to the parental molecule and each containing one parental strand + one new strand — semi-conservative.

DNA polymerase has a proofreading activity: if it inserts the wrong base, it can reverse and replace it. This reduces the error rate to ~1 per 10⁹ bases. Even so, some mutations slip through and become the basis of genetic variation and disease.

When Watson and Crick proposed the double helix in 1953, they suggested it could replicate by separating the two strands. Three models were possible:

• Semi-conservative — each daughter has 1 old + 1 new strand.
• Conservative — both old strands stay together in one daughter; both new strands together in the other.
• Dispersive — old and new pieces are scattered along each strand of each daughter.

In 1958 Meselson and Stahl distinguished between these models using the nitrogen isotopes ¹⁴N (light) and ¹⁵N (heavy):

1. Grow E. coli for many generations in ¹⁵N medium → all DNA bases contain heavy nitrogen → DNA is fully 'heavy'.
2. Transfer cells to ¹⁴N medium → any new DNA strand made will be 'light'.
3. Extract DNA at successive generations and run it on a caesium chloride density gradient. DNA forms a band at the position matching its density.

Generation 0 (parent, ¹⁵N): one band at heavy position.

Generation 1: a single band at intermediate density. This means every DNA molecule has the same density — half heavy, half light.

• This is exactly what semi-conservative predicts (one old ¹⁵N strand + one new ¹⁴N strand per molecule).
• It is not what conservative predicts (which would have given two bands: heavy + light).
• It is consistent with dispersive (which would also give an intermediate band).

Generation 2: two bands — one at intermediate density and one at fully light density.

• This matches semi-conservative: half the molecules have 1 old + 1 new = intermediate; half have 2 new strands = light.
• This is not what dispersive predicts (which would have given one band, gradually getting lighter).

The combined gen 1 + gen 2 results therefore eliminated both alternative models, leaving semi-conservative as the only explanation. Meselson-Stahl is often called the 'most beautiful experiment in biology' because of how cleanly it tested three competing hypotheses.

Although DNA polymerase has a proofreading function, occasional errors slip through and become mutations — permanent random changes in the base sequence. Mutations are the raw material of evolution and underlie many genetic disorders.

The three classes of point mutation are:

1. Substitution — one base is replaced by another. There are three sub-types based on effect on the protein:
   • Silent: new codon codes for the same amino acid (because the genetic code is degenerate) → no change in protein.
   • Missense: new codon codes for a different amino acid → primary structure changes, may alter tertiary structure and function.
   • Nonsense: new codon is a STOP codon → polypeptide is truncated, almost always non-functional.
2. Insertion — an extra base is inserted. This causes a frameshift: every codon downstream of the insertion is shifted, so the amino acid sequence from that point onwards is completely different. The protein is usually non-functional.
3. Deletion — a base is removed. This also causes a frameshift with the same consequences as insertion.

If the mutation is in or near the part of the gene that codes for the active site of an enzyme, the enzyme may lose its function. If the mutation is in a non-coding region or in a redundant codon position (e.g. the third base), it may have little or no effect.

Sickle-cell disease is the classic example: a single base substitution (A → T) in the β-globin gene changes one amino acid (glutamic acid → valine) and alters the haemoglobin tertiary structure, causing red blood cells to sickle under low oxygen.