CHAPTER 14

A Modern Understanding of DNA

Objectives

• Describe key experiments that helped identify that DNA is the genetic material.

• Explain transformation of DNA.

• State and explain Chargaff's rules.

• Describe the structure of DNA.

• Discuss similarities and differences between eukaryotic and prokaryotic DNA.

• Explain the Sanger method of DNA sequencing.

• Describe Meselson and Stahl experiments.

• Discuss the role of different enzymes and proteins in supporting DNA replication.

• Explain the process of DNA replication in prokaryotes.

• Discuss similarities and differences between DNA replication in eukaryotes and prokaryotes.

• State the role of enzymes in eukaryotic DNA replication.

• Discuss different types of mutations in DNA.

• Explain DNA repair mechanisms.

DNA: Deoxyribonucleic Acid

• Retrievability: DNA can be retrieved from hair, blood, or saliva.

• Uniqueness: Allows for identification because each person's DNA is unique.

• Applications of DNA Analysis:

  • Determining paternity

  • Tracing genealogy

  • Identifying pathogens

  • Archeological research

  • Tracing disease outbreaks

  • Studying human migration patterns

  • DNA diagnostics

  • New vaccine development

  • Cancer therapy

• Chromosome Composition in Humans: 23 pairs of chromosomes (one set from each parent).

• Mitochondrial DNA: Inherited directly from the female parent, which can involve inherited genetic disorders.

• Gene Mapping: Thousands of genes on each chromosome determine genotype and phenotype.

• Definition of Gene: A region of DNA that codes for a protein or another functional product.

• Haploid Genome Details: Has three billion base pairs and approximately 20,000–25,000 functional genes.

Historical Context

Discovery of DNA

• Understanding of DNA began with the discovery of nucleic acids leading to the double-helix model.

• Friedrich Miescher (1860s) isolated nuclein (later known as DNA) from white blood cells, a phosphate-rich chemical.

Key Experiments Identifying DNA as Genetic Material

• Frederick Griffith (1928) - Bacterial Transformation:

  • Transformation: Process by which a prokaryote takes in DNA shed by other prokaryotes.

  • Experiment with Streptococcus pneumoniae injected into mice:

  • Rough strain (R) - nonpathogenic

  • Smooth strain (S) - pathogenic with a polysaccharide capsule.

  • Results:

  • Mice injected with living S strain died from pneumonia.

  • Mice injected with living R strain survived.

  • Mice injected with heat-killed S strain survived.

  • Mice injected with a mixture of living R strain and heat-killed S strain died, recovering the S strain.

  • Conclusion: A transforming principle passed from heat-killed S strain to living R strain, converting it to pathogenic.

• Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944):

  • Isolated components from the S strain and transformed the R strain.

  • Results: Transformation did not occur when DNA was degraded, confirming DNA as the transforming principle.

• Martha Chase and Alfred Hershey (1952):

  • Demonstrated that DNA, not proteins, is the genetic material using bacteriophages.

  • Bacteriophage: A virus that infects bacteria, consisting of a protein coat and nucleic acid core (DNA or RNA).

  • Method used radioactive elements to distinguish between protein and DNA in cells:

  • Radioactive sulfur (35S) tagged proteins.

  • Radioactive phosphorus (32P) tagged DNA.

  • Results:

  • In 35S phage, radioactive signal found in the supernatant (indicating proteins did not enter bacterial cells).

  • In 32P phase, signal found in the pellet with bacterial cells, confirming that DNA was injected into host cells.

• Erwin Chargaff:

  • Found species have different amounts of adenine (A), thymine (T), guanine (G), and cytosine (C).

  • Pattern within individuals showed that:

  • A = T (Chargaff's First Rule)

  • C = G (Chargaff's Second Rule)

  • Resulted in the understanding of base pairing which informed Watson and Crick's double helix model.

Chargaff's Rules

• Example rule breakdown: If a strand of DNA contains 15% adenine, it will have 15% thymine.

• Remaining percentage constituted guanine and cytosine, with specific ratios guided by Chargaff's rules.

Structure of DNA

Nucleotide Composition

• Nucleotides, the building blocks of DNA and RNA, consist of:

  • Nitrogenous base

  • 5-carbon sugar (deoxyribose for DNA, ribose for RNA)

  • Phosphate group

• Nucleotides can be purines (A, G) or pyrimidines (C, T, U).

Phosphodiester Bond Formation

• Carbons of the sugar are numbered 1', 2', 3', 4', and 5'.

  • Phosphate attached to the 5' carbon, with 3' carbon connected to a hydroxyl group; linkages create strands.

  • Formation of 3'-5' phosphodiester bonds between nucleotides creates a sugar-phosphate backbone.

DNA Structural Model

• Watson and Crick determined DNA structure (1950s); used Rosalind Franklin’s X-ray diffraction data.

• Confirmed DNA is composed of two strands twisted in a right-handed helix.

• A pairs with T, C pairs with G, stabilized by hydrogen bonds; A-T pairs form 2 hydrogen bonds, C-G pairs form 3 hydrogen bonds.

• Strands are antiparallel (3’ end facing 5’ end), with specific distances between base pairs and the helical structure.

• Structural details:

  • Each turn of the helix measures 3.4 nm, with 10 base pairs per turn.

  • Uniform diameter of 2 nm with major and minor grooves formed by sugar-phosphate backbones.

DNA Sequencing

• Fred Sanger (Dideoxy Chain Termination Method):

  • Used in sequencing the human genome.

  • Method involves dideoxynucleotides (ddNTPs) to terminate DNA strands, allowing distinction of sequence based on lengths of fragments.

• Process involves denaturing DNA, synthesizing with DNA polymerase, and using fluorescently labeled ddNTPs.

DNA Replication Processes

Prokaryotic DNA Replication

• Initiation occurs at origin of replication; follows the semiconservative model.

• Processes: 1) Helicase unwinds DNA, 2) Primase synthesizes RNA primer, 3) DNA pol III adds nucleotides.

• Structural differences include leading vs. lagging strands:

  • Leading strand synthesized continuously, lagging strand formed in Okazaki fragments.

  • DNA ligase seals fragments into continuous DNA.

Enzymes in DNA Replication

• Key enzymes include:

  • Helicase: Unwinds DNA.

  • Primase: Synthesizes RNA primers.

  • DNA polymerases: Synthesizes new DNA (pol III as main polymerase).

  • Ligase: Seals gaps between Okazaki fragments.

Differences in Eukaryotic and Prokaryotic DNA Replication

• Eukaryotic replication has multiple origins, runs slower (approximately 50-100 nucleotides/sec compared to 1,000 nucleotides/sec in prokaryotes), and involves more proteins.

• Eukaryotic DNA is packaged with histone proteins; whereas prokaryotic DNA is less complex.

Mutations in DNA

• Types of Mutations:

  • Point mutations, frameshift mutations (insertions/deletions), translocations, and triplet repeats.

• Induced mutations: Result from environmental factors (UV rays, chemicals).

• Spontaneous mutations: Result from internal biological processes.

• Mutations in repair genes can lead to disorders such as skin cancer or hereditary hemophilia.

DNA Repair Mechanisms

• Mechanisms Include:

  • Proofreading by DNA polymerase

  • Mismatch repair involving proper base pairing assessments post-replication.

  • Nucleotide Excision Repair, useful for correcting thymine dimers from UV exposure.

Summary of Key Concepts

• DNA structure ranges from single circular chromosomes in prokaryotes to complex linear arrangements in eukaryotes.

• Replication processes underline DNA's fidelity through proofreading mechanisms but are still subject to mutations.

• The understanding and manipulation of DNA underpin advancements in genetics, forensic science, and medical research.