Molecular Genetics: DNA Structure, Replication, and Repair

Posted on Nov 22, 2025 in Biology

DNA Structure and Packaging

  • Phosphate Group: Gives DNA its acidic properties.
  • DNA Orientation: Written in the 5′ to 3′ direction.
  • Complementary Pairing: Purine pairs with pyrimidine (A-T, G-C) ensuring constant width.
    • Adenine (Purine) pairs with Thymine (Pyrimidine) via 2 hydrogen bonds.
    • Guanine (Purine) pairs with Cytosine (Pyrimidine) via 3 hydrogen bonds.
  • Backbone: Antiparallel structure; sugars are oriented differently to allow base pairing. The helix shields hydrophobic bases. There are approximately 10 base pairs per helical turn.
  • Packaging: Approximately 3 billion base pairs, totaling about 2 meters of DNA.
  • Chromosomes: 23 pairs in humans. They contain the same genes but differ in inherited sequence (alleles, leading to different traits). Formed by condensed chromatin fiber loops.
  • Histones: Proteins that allow the wrapping of DNA. They are rich in positively charged Lysine and Arginine residues, which bind strongly with the negatively charged DNA phosphate backbone.
  • Nucleosome: The fundamental unit of chromatin, consisting of a histone/DNA complex (DNA wrapped around an octamer of 8 histone proteins).
  • Chromatin: Nucleosomes packed on top of one another. This level of packing is maintained unless the cell enters mitosis.
  • Chromatin Remodeling Complexes: Use ATP energy to loosen DNA and push it along the histone octamer, allowing access to genes (activation). They can also make DNA less accessible (gene regulation).

The Process of DNA Replication

Synthesis Direction
New nucleotides are always added in the 5′ to 3′ direction.
Origin of Replication (Ori)
The starting point of replication. It contains an abundance of A-T pairs (easier to pull apart due to only 2 H-bonds). Eukaryotes contain multiple origins that merge; prokaryotes typically contain one.
Helicase
Unwinds the DNA double helix and breaks the hydrogen bonds.
Single-Strand Binding Proteins
Bind to the separated strands to prevent hydrogen bonds from re-forming.
Topoisomerase
Relieves torsional stress (supercoiling) by creating and resealing temporary single-strand breaks ahead of the replication fork.
Primase
Synthesizes short RNA primers, providing DNA Polymerase with a necessary starting point (a free 3′-OH group).
DNA Polymerase
Reads the template strand from 3′ to 5′ but synthesizes the new strand from 5′ to 3′. It catalyzes the addition of nucleotides. Energy for this reaction comes from the hydrolysis of the phosphate bond in the incoming nucleoside triphosphate. It possesses both a Polymerization site and an Editing site (for proofreading mistakes at the 3′ end).
Lagging Strand Synthesis
Requires continuous addition of RNA primers by Primase. DNA Polymerase synthesizes the new strand discontinuously, creating small fragments called Okazaki fragments.

Post-Replication Processing

  • Nuclease: Removes the RNA primers.
  • DNA Repair Polymerase: Fills the resulting gaps with complementary DNA.
  • DNA Ligase: Seals the remaining nicks (gaps in the sugar-phosphate backbone).
  • Telomerase: Addresses the gap left by the primer on the lagging strand ends (telomeres). It contains an RNA sequence that complements the 3′ overhang, using this sequence to synthesize repetitive, non-coding DNA on the overhang. This extended DNA then serves as a template region for Primase and DNA Polymerase to synthesize the missing gap DNA.

Historical Discoveries in Genetics

Early Belief
The original belief was that proteins carried genetic material because of their greater complexity (20 amino acids versus 4 nucleotides).
Bacteriophage T2
A virus that specifically infects and replicates within bacteria. Its genetic material is contained in the viral head (nucleocapsid). It injects this material into the bacterial cell to produce new phages.

Key Experiments Proving DNA is the Genetic Material

  1. Griffith’s Transformation Experiment (1928)

    Used Streptococcus pneumoniae (Smooth ‘S’ strain, which has a sugar capsule and is virulent; Rough ‘R’ strain, which lacks a capsule and is non-virulent).

    • S strain killed the mouse.
    • R strain did not kill the mouse.
    • Heat-killed S strain did not kill the mouse.
    • Living R strain mixed with heat-killed S strain killed the mouse.

    Conclusion: A chemical substance from the dead S cells was capable of being transferred to and transforming the living R cells.

  2. Avery, MacLeod, & McCarty Experiment (1944)

    Took dead S strain extract and treated separate vials with either RNase, Protease, or DNase. They incubated these vials with R strain bacteria and observed the outcome.

    The vial treated with DNase did not produce the virulent S strain (and did not kill mice).

    Conclusion: DNA is the chemical substance responsible for converting the R strain to the S strain.

  3. Hershey-Chase Experiment (1952)

    Used E. coli incubated with Bacteriophage T2 labeled with either radioactive Phosphorus-32 ($ ext{^{32}P}$, labels DNA) or Sulfur-35 ($ ext{^{35}S}$, labels protein). The labeled phages infected E. coli. After agitation to remove phages on the surface, the bacteria were pelleted and analyzed.

    Result: $ ext{^{32}P}$ (DNA) was found in the bacterial pellet; $ ext{^{35}S}$ (protein) was found in the supernatant fluid.

    Conclusion: DNA is the substance responsible for phage T2 replication.

DNA Replication Models and Proof

Watson & Crick (1953)
Discovered the double helix structure of DNA.
Semiconservative Replication
Each new DNA molecule consists of one new strand and one original template strand.
Conservative Replication
The original molecule is preserved entirely, and a completely new molecule is synthesized.
Dispersive Replication
Both new DNA molecules contain interspersed regions of new and old DNA on both strands.
Meselson-Stahl Experiment (1958)
Grew E. coli in heavy Nitrogen ($ ext{^{15}N}$) medium, then transferred them to light Nitrogen ($ ext{^{14}N}$) medium. DNA was extracted and separated by density ultracentrifugation.
  • After Round 1: Only one intermediate density band was observed, ruling out the Conservative model.
  • After Round 2: Two bands were observed (one intermediate, one light), ruling out the Dispersive model.

Conclusion: DNA replicates semi-conservatively, where each strand serves as a template for a new strand through complementary base pairing.

SARS-CoV-2 (COVID-19) Genome

The SARS-CoV-2 genome is a single-stranded RNA molecule, approximately 30,000 nucleotides long. It folds over itself to form hydrogen bonds, protecting the hydrophobic nitrogenous bases.

  • Genome Organization:
    • The first two-thirds of the genome encode replicating genes, translated by host ribosomes.
    • The last one-third of the genome encode structural genes, translated by the virus’s own machinery or specific ribosomal frameshifting.
  • Key Viral Enzymes:
    • 3CL-Protease: A cysteine protease that cleaves the large viral polypeptide chain into functional proteins.
    • Helicase: Unwinds the secondary structure within the RNA genome.
    • RNA-dependent RNA Polymerase (RdRp): Replicates the entire genome by first producing a complementary negative-sense RNA strand, which is then used as a template to replicate new positive-sense viral genomes.
  • Cell Entry Methods: The virus enters the host cell via membrane fusion or endocytosis.

Mechanisms of DNA Repair

Consequences and Causes of DNA Damage

  • Genetic Changes: Small genetic changes can be beneficial, or have no effect if they occur in non-coding regions or if codon redundancy prevents a change in the amino acid sequence. However, they can cause diseases like cancer or inheritable conditions, such as Cystic Fibrosis (CF), which involves the loss of three nucleotides encoding phenylalanine in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.
  • Endogenous Damage: Caused by reactive chemicals produced during metabolism (e.g., hydrogen peroxide) or spontaneous chemical reactions (e.g., base modification).
  • Environmental Factors: Include UV radiation, ionizing radiation, and toxic chemicals.

Common Types of DNA Damage

Depurination
The removal of Guanine (G) or Adenine (A) bases from the DNA backbone.
Deamination
The removal of an amino group, such as Cytosine (C) being deaminated to produce Uracil (U).
Thymine Dimer
Forms when UV light is absorbed, causing two adjacent thymine bases to covalently link. This causes distortion in the backbone, stalls DNA Polymerase, and is recognized by repair proteins.

Repair Pathways

The undamaged strand always acts as the source of information to restore the damaged strand, as it is easily distinguished from the damaged region.

Basic Excision Repair (Base or Nucleotide Excision)

  1. The damaged base or nucleotide is recognized and removed by nucleases.
  2. Repair DNA Polymerase binds to the 3′ end of the cut DNA and fills in the missing nucleotides using the undamaged strand as a template.
  3. The remaining nick is sealed by DNA Ligase.

Mismatch Repair (MMR)

Occurs immediately after DNA replication. The newly synthesized strand is distinguished from the template strand, often because of transient nicks present in the new strand.

Double-Strand Break Repair

These are highly dangerous breaks that sever both strands of the DNA helix.

Nonhomologous End Joining (NHEJ)
Used when the cell has no mechanism (like a sister chromatid) to replace the lost information. It quickly repairs the break before the DNA fragments drift apart. Ends are cleaned up by nucleases and joined by ligase, resulting in a loss of nucleotides at the repair site (error-prone).
Homologous Recombination (HR)
Occurs shortly after DNA replication when a sister chromatid is available to serve as a perfect template for repair (accurate).
  1. The broken 3′ end invades the homologous DNA duplex and searches for the complementary sequence.
  2. The invading strand is elongated by DNA Repair Polymerase.
  3. The invading strand is released, and DNA synthesis continues using the complementary strand from the damaged DNA as a template.
  4. The break is accurately repaired with no loss of genetic information.