DNA Structure and Genome Organisation

Structure of DNA:

B-Form DNA:

• Right-handed double helix.

  • There are ~10.4 base pairs per helical turn, with a helix diameter of ~2nm.

• Bases are stacked almost flat, separated by ~0.34nm.

• Strands are antiparallel (5’→3’ vs 3’→5’).

Grooves:

• The major groove (~2.2nm wide) is rich in chemical information.

  • Base pairs present unique hydrogen bond donor and acceptor patterns.

    • E.g. a G-C pair exposes a different pattern in the major groove than an A-T pair would.

      • Transcription factors and DNA-binding proteins exploit this for sequence specificity.

• The minor groove (~1.2nm wide) is narrower and has less chemical information.

  • It is recognised mainly by small molecules (e.g. some antibiotics).

Structural Importance Beyond Sequence:

• DNA is not just a passive information store; its 3D structure guides how proteins recognise it.

• Local flexibility and groove accessibility influence replication initiation, repair enzyme recognition and transcription factor binding.

Alternative DNA Forms:

A-Form:

• Right handed, shorter and wider helix.

  • Appears under dehydrated conditions, or in DNA-RNA hybrids (like replication primers).

  • There are ~11bp per turn.

  • More compact, bases tilted relative to axis.

    • The tilting changes groove architecture, narrowing the major groove and widening the minor groove (as bases are usually perpendicular to the axis to maximise π-π overlap).

      • ❗ The altered groove geometry of A-form helices facilitates primer synthesis because the shallow minor groove makes DNA–RNA hybrids accessible to RNA-binding proteins. This is essential for stable primer formation during replication, but replication overall still depends on B-form DNA as the main template structure.

Z-Form:

• Left handed, zig-zag backbone.

  • Favoured in GC-rich or alternating purine-pyrimidine sequences.

  • Can form transiently during transcription and is thought to play roles in gene regulation and recombination.

Local Distortions and Bends:

• A-tracks (stretches of adenine) cause intrinsic bending.

  • These bends are important for DNA packaging in nucleosomes and in forming promoter structures accessible to RNA polymerase.

    • Bending can be protein-induced (e.g. TATA-binding protein clamps DNA open at promoters).

Recognition is often a combination of direct readout (hydrogen bonds in major groove) and indirect readout (DNA shape and bendability).

Protein-DNA Interactions:

Charge Complementarity:

• The DNA phosphate backbone has a negative charge.

• DNA-binding proteins often have basic domains (arginine, lysine-rich).

  • E.g. histones have highly basic tails.

Sequence Specificity:

• Proteins use hydrogen bonds, Van der Waals, and hydrophobic contacts.

  • E.g. helix-turn-helix proteins (like the lac repressor) insert an alpha helix into the major groove to read base pairs.

DNA Distortion by Proteins:

• Binding is not always passive. Proteins can bend, kink, or unwind DNA to facilitate replication or transcription.

  • E.g. DNA polymerase bends DNA 90 degrees in its active site to separate the template strand for copying.

DNA Supercoiling and Topology:

Supercoiling:

• When DNA is under- or over-wound, it compensates by forming superhelical twists (writhe).

  • Negative Supercoils - underwound DNA, promotes strand separation and is useful for replication and transcription.

  • Positive Supercoils - overwound DNA, forms ahead of replication forks and blocks progress if not removed.

Enzymes:

• Topoisomerase I - makes single-strand nicks, and relaxes supercoils passively.

• Topoisomerase II (DNA Gyrase in bacteria) - cuts both strands, passes another duplex through, and actively introduces negative supercoils using ATP.

• Eukaryotes lack gyrase but rely on Topoisomerase I/II to manage torsional stress.

DNA Replication:

DNA Polymerases extend in the 5’ to 3’ direction, and a primer with a free 3’-OH is needed.

Initiation in E.Coli:

• Origin (oriC) - 245 base pairs with AT-rich repeats (easier to unwind).

• DnaA - initiator protein - binds to oriC and melts DNA locally with ATP hydrolysis.

• DnaB (helicase) - loaded by DnaC, and unwinds the duplex.

• Primase (DnaG) - synthesises short RNA primers.

Elongation:

• Leading Strand - continuous synthesis in the direction of the replication fork.

• Lagging Strand - discontinuous synthesis as Okazaki fragments.

• DNA Pol III Holoenzyme - main polymerase, highly processive because of the β-clamp sliding ring.

• SSBs (Single-Stranded Binding Proteins) - stabilises unwound DNA.

• Topoisomerases - prevents supercoiling ahead of the replication fork.

Maturation of Okazaki Fragments:

• DNA Pol I - removes RNA primers via 5’→3’ exonuclease, and fills gaps with DNA.

• DNA Ligase - seals nicks (ATP- or NAD⁺-dependent).

Coordination:

• The lagging strand loops so both polymerases move in the same physical direction.

  • This is known as the “trombone model”.

❗ Primase is needed because DNA polymerases cannot start de novo synthesis.

Replication Topology:

• As the fork moves, it introduces positive supercoils ahead.

  • Without topoisomerases, DNA would quickly become overwound and stall.

    • Topoisomerase II solves this by transiently cutting both strands and passing another duplex through.

Genome Organisation:

Non-Coding DNA:

• Microsatellites: short tandem repeats that are unstable; used in DNA fingerprinting.

• Transposons:

  • Class I (Retrotransposons): “copy-paste” via RNA intermediates.

  • Class II (DNA Transposons): “cut-paste” directly via transposase enzyme.

• Pseudogenes: inactivated genes, often from duplication followed by mutation.

• Regulatory DNA: enhancers, silences, insulators. They are crucial for control.
  

Gene Families:

• Multiple related genes cluster together (globins, keratins, olfactory receptors etc.).

• Allows functional specialisation (e.g. foetal vs adult haemoglobin).

An open reading frame (ORF) is a stretch of DNA that can be translated into a protein without interruption by a stop codon.

• It starts with a start codon (AUG in mRNA, ATG in DNA).

• It continues in triplet codons that each specify an amino acid.

• It ends only when a stop codon (UAA, UAG, or UGA) is reached.

Over time, pseudogenes accumulate point mutations (single-base changes) and indels (insertions or deletions).

• These can disrupt the original ORF by:

  • Introducing a premature stop codon.

  • Causing a frameshift (changing the reading frame).

  • Altering essential splice sites or promoter regions.

• Once the ORF is disrupted, the gene can’t produce a proper protein, even if it’s still transcribed.