Enzymes, DNA Replication, and Genetic Material Discovery

Enzymes: Biological Catalysts

Enzymes are globular proteins that act as catalysts for biochemical reactions occurring inside or outside the cell.

Enzyme Composition

There are two main types of enzymes based on composition:

• Holoproteins: Consist solely of an amino acid chain (e.g., ribonuclease, lysozyme).
• Heteroproteins: Composed of two components: an apoenzyme (the protein part) and a cofactor (a non-protein part, which can be a coenzyme or a prosthetic group).

Factors Affecting Enzyme Activity

Enzyme activity is influenced by factors such as temperature (T°) and the concentration of the substrate.

Enzyme Inhibitors

Enzyme activity can be reduced or stopped by inhibitors, which can be:

• Competitive Inhibitors
• Noncompetitive Inhibitors

Enzyme Classification

Enzymes are classified based on the type of reaction they catalyze:

• Oxidoreductases: Catalyze oxidation-reduction reactions.
• Transferases: Transfer functional groups between molecules.
• Hydrolases: Catalyze hydrolysis (often digestive enzymes).
• Lyases: Cleave chemical bonds without hydrolysis or oxidation.
• Isomerases: Catalyze the rearrangement of atoms within a molecule.
• Ligases (or Synthetases): Join two molecules together, usually requiring energy (e.g., from ATP).

DNA Replication Process

DNA replication (or duplication) is the process by which the genetic information stored in DNA is accurately copied.

Discovery of DNA as Genetic Material

In 1953, James Watson and Francis Crick established the double-helix model of the DNA molecule, which made clear how DNA could function as the genetic material. DNA contains coded information within its sequence of nitrogenous bases.

Key Components in Replication

Several molecules and enzymes are crucial for DNA replication:

• DNA Molecule (Template): Serves as the pattern for synthesizing the new DNA strands.
• DNA Helicases: Unwind the parental DNA double helix at the replication fork.
• Single-Strand Binding Proteins (SSBPs): Bind to the separated DNA strands, keeping them from re-annealing and protecting them.
• Topoisomerases: Relieve the torsional stress generated by DNA unwinding by breaking and rejoining DNA strands ahead of the replication fork.
• Deoxyribonucleotide Triphosphates (dNTPs): The building blocks (A, T, C, G) used to synthesize the new DNA strands; the cleavage of their phosphate bonds provides energy for polymerization.
• DNA Polymerase: Synthesizes new DNA strands by adding nucleotides complementary to the template strand, always in the 5' to 3' direction (adding to the free 3'-OH group).
• Primase: An RNA polymerase that synthesizes short RNA primers, providing a starting point (a free 3'-OH group) for DNA polymerase.
• RNA Nucleotides: Used by primase to construct the RNA primers.
• DNA Ligase: Joins the Okazaki fragments on the lagging strand and seals other nicks in the DNA backbone.

Historical Experiments Identifying DNA

Mendel's Work on Heredity (1865)

In 1865, Gregor Mendel established the fundamental principles of heredity through his experiments with pea plants, laying the groundwork for genetics.

Griffith's Transformation Experiment (1928)

In 1928, Frederick Griffith conducted experiments on bacterial transformation using two strains of Streptococcus pneumoniae bacteria and mice. The virulent 'S' strain (smooth, encapsulated) caused lethal pneumonia, while the non-virulent 'R' strain (rough, non-encapsulated) did not. Griffith found that injecting mice with a mixture of heat-killed 'S' strain bacteria and live 'R' strain bacteria resulted in the death of the mice, and live 'S' strain bacteria could be recovered from them. This indicated that some component from the dead 'S' strain bacteria had transformed the live 'R' strain bacteria into the virulent 'S' strain – he called this the 'transforming principle'.

Alloway's In Vitro Transformation (1933)

In 1933, J. Lionel Alloway demonstrated that transformation could occur *in vitro* (in a test tube). He prepared cell-free extracts from heat-killed 'S' strain bacteria and showed that mixing these extracts with live 'R' strain bacteria could transform them into the 'S' strain, confirming Griffith's findings without using mice.

Avery, MacLeod, and McCarty Experiment (1944)

In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty systematically purified the transforming principle from 'S' strain bacteria. Through careful experimentation, including the use of enzymes that degrade specific macromolecules (proteases, RNases, DNases), they demonstrated conclusively that DNA, and not protein or RNA, was the molecule responsible for transformation. They isolated DNA from the 'S' strain, mixed it with live 'R' strain bacteria, and observed the transformation into live, virulent 'S' strain bacteria. Treatment of the extract with DNase abolished its transforming ability, while treatments with protease or RNase did not.

Bacteriophages: Viruses Infecting Bacteria

Bacteriophages (often shortened to phages) are viruses that specifically infect bacteria. The name derives from Greek, meaning "bacteria eaters". A bacteriophage is entirely dependent on its host bacterium for all aspects of its replication and life cycle.