DNA as the Genetic Material

Characteristics of Hereditary Material

The identification of DNA as the genetic material was based on several key properties required for heredity:

• Localization: Found in the nucleus and as a component of chromosomes.

• Stability: Present in a stable form within cells.

• Information Content: Contains instructions for structure, function, development, and reproduction.

• Accurate Replication: Ensures daughter cells inherit identical information.

• Mutability: Low mutation rate allows for genetic variation and evolution.

Historical Timeline of Discoveries

Major milestones in the discovery of DNA as the genetic material:

Key Experiments

• Griffith (1928): Demonstrated transformation in bacteria, suggesting a hereditary molecule.

• Avery, MacLeod & McCarty (1944): Showed DNA is the transforming factor by selectively destroying DNA, RNA, protein, or lipids/polysaccharides.

• Hershey–Chase (1952): Used radioactive isotopes to show DNA, not protein, is inherited in bacteriophages.

Nucleotide and Polynucleotide Structure

Nucleotide Components

A nucleotide is the basic unit of DNA and RNA, consisting of:

• Phosphate group

• 5-carbon sugar: Deoxyribose (DNA) or ribose (RNA)

• Nitrogenous base: Purines (Adenine, Guanine), Pyrimidines (Thymine, Cytosine in DNA; Uracil, Cytosine in RNA)

Nucleoside: Sugar + base (no phosphate). Nucleotide: Nucleoside + phosphate.

Polynucleotide Structure

• Phosphodiester bond: Covalent bond joining the 3′ OH of one nucleotide to the 5′ phosphate of the next.

• Polarity: DNA strands have a 5′ end and a 3′ end.

Solving the Structure of DNA

Evidence for the Double Helix

• Chargaff's Rules: The amount of adenine equals thymine, and guanine equals cytosine.

• X-ray Diffraction: Rosalind Franklin's Photo 51 revealed DNA's helical structure and dimensions.

• Model Building: Watson & Crick used available data to propose the double helix model.

Features of the DNA Double Helix

Structural Properties

• Antiparallel Strands: Two polynucleotide strands run in opposite directions.

• Base Pairing: A–T pairs via 2 hydrogen bonds; G–C pairs via 3 hydrogen bonds.

• Dimensions: One turn every 10 base pairs (3.4 nm); base pairs are stacked 0.34 nm apart.

• Major and Minor Grooves: Created by base-pair stacking; sites for protein binding.

• Structural Forms: DNA can adopt A, B, or Z forms depending on conditions.

Overview of DNA Replication

General Mechanism

• Each parental strand remains intact and serves as a template.

• Replication produces two identical daughter duplexes, each with one parental and one new strand.

Models of Replication

• Conservative: Parental strands stay together; new strands form a separate duplex.

• Semiconservative: Each duplex contains one parental and one new strand.

• Dispersive: Parental and new DNA are interspersed in both strands.

Meselson–Stahl Experiment: Used isotopic labeling to distinguish models; supported semiconservative replication.

Where Replication Begins

Origins and Replication Forks

• John Cairns (1963): Used autoradiography to visualize replicating E. coli chromosomes, revealing theta (θ) structures.

• Replication Fork: Site where DNA strands are separated for synthesis.

• Bidirectional Replication: Two forks move away from the origin in opposite directions.

• Eukaryotes: Multiple origins; thousands per genome.

Initiation of Replication in E. coli

Order of Events

1. DnaA: Binds 9-mer, bends DNA, breaks H-bonds in A-T-rich 13-mer.

2. DnaB (helicase): Unwinds DNA; DnaC delivers DnaB.

3. SSB: Prevents reannealing of separated strands.

4. Topoisomerases: Relieve torsional stress and prevent supercoiling.

The Replication Fork

Enzymes and Mechanisms

• Primase: Synthesizes RNA primers for DNA polymerase.

• DNA Polymerase III: Main enzyme for DNA synthesis; requires a sliding clamp for processivity.

• DNA Polymerase I: Removes RNA primers (5′→3′ exonuclease) and fills gaps (5′→3′ polymerase).

• DNA Ligase: Seals nicks between fragments.

• Proofreading: 3′→5′ exonuclease activity corrects errors.

The End Replication Problem and Telomeres

Problem and Solution

• End Replication Problem: Lagging strand cannot be fully replicated at the chromosome end, leading to shortening.

• Telomeres: Repetitive DNA sequences (e.g., TTAGGG in humans) protect chromosome ends.

• Telomerase: Ribonucleoprotein enzyme extends telomeres using its RNA template; active in germ-line and some stem cells.

• Telomere Length: Important for chromosome stability, cell longevity, and reproductive success.

Applications of DNA Replication

PCR (Polymerase Chain Reaction)

PCR is an automated method to amplify DNA, requiring knowledge of the target sequence for primer design.

• Limitations: Products longer than ~10–15 kb are difficult; Taq polymerase lacks proofreading.

• Applications: Genotyping, ancient DNA analysis.

Sanger (Dideoxy) Sequencing

• Uses DNA polymerase and dNTPs, plus small amounts of ddNTPs (lacking 3′-OH).

• Incorporation of ddNTP terminates synthesis.

• Automated sequencing uses fluorescently labeled ddNTPs.

Next-Generation Sequencing (Illumina)

• Genome fragmented; fragments attached to flow cell via adaptors.

• Fragments amplified; sequencing uses reversible terminating fluorescent nucleotides.

• High throughput: millions of fragments sequenced simultaneously.

Quick-Review Checklist

• Describe key experiments (Griffith, Avery, Hershey–Chase).

• Draw and label a nucleotide; name purines and pyrimidines.

• State double-helix dimensions and base-pairing hydrogen bond counts.

• Compare A-, B-, and Z-DNA.

• Explain replication models and Meselson–Stahl experiment.

• Order E. coli initiation proteins: DnaA → DnaB/DnaC → SSB → topoisomerase.

• Explain lagging strand synthesis and roles of pol III, pol I, primase, ligase.

• Describe the end replication problem and telomerase function.

• Describe PCR steps and Sanger sequencing mechanism.

Key Equations and Concepts

Chargaff's Rule

For double-stranded DNA:

Phosphodiester Bond Formation

Formation between nucleotides:

PCR Cycle

Each cycle doubles the DNA:

Sanger Sequencing Termination

ddNTP lacks 3′-OH, preventing further elongation:

Practice Questions and Answers

1. Avery's DNase-treated extract failed to transform rough bacteria. What does that show? Answer: DNA is the transforming factor.

2. Why did Hershey and Chase label DNA with ³²P and protein with ³⁵S? Answer: DNA contains phosphorus but no sulfur; protein contains sulfur but almost no phosphorus.

3. A DNA sample is 30% adenine. What percent is guanine? Answer: 20% (A=T=30%, G=C=20%).

4. Why can the leading strand be copied to the end of a linear chromosome but the lagging strand cannot? Answer: The lagging strand needs RNA primers, and the last one cannot be replaced.

5. A polymerase adds a wrong base. Which activity fixes it, and in which direction does it work? Answer: 3′→5′ exonuclease proofreading.

6. Why must PCR use a heat-stable polymerase? Answer: It must survive the 95 °C denaturation steps.

7. Why does a ddNTP stop chain elongation? Answer: It lacks the 3′-OH needed to form the next phosphodiester bond.

8. Why does E. coli only need ~33 minutes to copy its genome? Answer: Replication is bidirectional from oriC, so two forks share the work.