DNA Structure, Replication, and Gene Mutations

Learning Outcomes for DNA Structure and Genetic Information

• Upon completion of this section, students should be able to:
  • Summarize the specific experiments that enabled scientists to determine that DNA constitutes genetic material.
  • Describe the detailed structure of a DNA molecule.
  • Describe the role of RNA molecules in gene expression.
  • Determine the sequence of amino acids in a peptide given a messenger RNA (mRNA) sequence.

Historical Timeline of DNA Discovery and Structural Analysis

• The determination of DNA as the carrier of genetic information occurred between 1928 and 1952.
  • Frederick Griffith (1928): Conducted early work suggesting nucleotide material could carry genetic information via bacterial transformation.
  • Alfred Hershey and Martha Chase (1952): Formally proved that nucleotides were the carriers of genetic information using bacteriophages.
• The structure of DNA was determined in the early 1960s based on findings from the late 1940s and 1950s.
  • Erwin Chargaff (late 1940s): Established "Chargaff’s Rules" regarding base proportions.
  • Rosalind Franklin (1952): Used X-ray diffraction to study DNA structure. Her "Image 51" was shown without her permission and provided critical data on the molecule's diameter and its two-strand nature.
  • James Watson and Francis Crick (1962): Awarded the Nobel Prize for determining the structure and functional implications of DNA.

Frederick Griffith’s Transformation Experiment (1928)

• Objective: Griffith was attempting to develop a vaccine for pneumonia.
• Experimental Organism: Streptococcus pneumoniae.
• Observed Strains:
  • S strain (Smooth): Appeared shiny under a microscope due to the presence of a mucus coat; this strain was deadly (pathogenic).
  • R strain (Rough): Appeared rough and lacked the mucus coat; this strain was not deadly (non-pathogenic).
• Experimental Process and Findings:
  • Injection of living S strain: Mouse dies.
  • Injection of living R strain: Mouse lives.
  • Injection of heat-killed S strain: Mouse lives (heat destroyed the ability of the S strain to cause disease).
  • Injection of a mixture of heat-killed S and living R strain: Mouse dies.
• Conclusion: Living S strain bacteria were recovered from the bodies of the dead mice. Griffith concluded that a "factor" had passed from the dead S strain to the living R strain, transforming the R strain into the deadly S strain.

The Hershey and Chase Experiment (1952)

• Core Question: Is genetic material composed of protein or nucleic acid?
• Experimental Model: Bacteriophages (viruses that infect bacteria).
  • The phage consists of a protein coat (capsid) and nucleic acid (DNA).
  • Phosphorus ($P$): Used to label nucleic acids, as phosphorus is present in DNA but not protein.
  • Sulfur ($S$): Used to label proteins, as sulfur is present in proteins but not DNA.
• Experiment 1 (DNA Labeling):
  • Phage DNA was labeled with radioactive phosphorus.
  • Labelled phages were allowed to infect bacteria.
  • The remains of the phage capsids were stripped away.
  • Radioactivity was found inside the bacterial cells, indicating DNA entered the host.
• Experiment 2 (Protein Labeling):
  • Phage protein was labeled with radioactive sulfur.
  • Labelled phages were allowed to infect bacteria.
  • The remains of the phage capsids were stripped away.
  • Radioactivity was found only in the surrounding liquid, not inside the bacteria.
• Conclusion: DNA, not protein, is the material transferred to bacteria to direct the production of new viruses.

Structural Fundamentals of DNA

• Erwin Chargaff’s Rules:
  • The proportion of Cytosine ($C$) to Guanine ($G$) and Adenine ($A$) to Thymine ($T$) remains constant across all known life.
  • Different species possess different overall proportions of $C$, $G$, $A$, and $T$.
• The Nucleotide Building Blocks: DNA is composed of four types of nucleotides, each consisting of a sugar, a phosphate group, and one of four nitrogenous bases.
  • Purine Bases (Double-ring structure): Adenine ($A$) and Guanine ($G$).
  • Pyrimidine Bases (Single-ring structure): Thymine ($T$) and Cytosine ($C$).
• Physical Shape: A double helix resembling a spiral staircase.
• Strand Composition:
  • The "backbone" of the strands consists of alternating sugar and phosphate groups.
  • Two strands of nucleotides run in opposite directions.
• Bonding and Pairing:
  • The two strands are held together by hydrogen bonds between the bases.
  • Complementary Base Pairing: Adenine always pairs with Thymine ($A-T$) and Guanine always pairs with Cytosine ($G-C$).
  • While base pairing is constant for all life on Earth, the specific biological information is encoded in the unique order of these bases.

The Mechanism of DNA Replication

• Basic Principle: Each of the two strands in the parent DNA molecule acts as a template for building a new complementary strand.
• Semiconservative Model: Proposed by Matthew Meselson and Franklin Stahl.
  • Each "daughter" DNA molecule consists of one original (parental) strand and one newly synthesized strand.
• Meselson and Stahl Experiment:
  • Labled "old" strands with a heavy isotope of nitrogen ($^{15}N$).
  • Labled "new" strands with a lighter isotope of nitrogen ($^{14}N$).
  • First Replication: Produced a single band of hybrid DNA, which eliminated the "conservative model."
  • Second Replication: Produced both light and hybrid DNA, which eliminated the "dispersive model" and confirmed the "semiconservative model."

Enzymatic Process of DNA Replication

• 1. Initiation:
  • Helicase: Binds to the origin of replication; it unwinds and opens the double helix.
  • Topoisomerase: Prevents the DNA from supercoiling (over-winding) ahead of the replication fork.
  • Single-Stranded Binding Proteins (SSBs): Bind to the exposed bases to keep the two strands separate.
• 2. Synthesis:
  • Primase: Adds RNA primers to both DNA strands to provide a starting point for synthesis.
  • DNA Polymerase: Binds to the primer and adds new DNA nucleotides. It catalyzes the covalent bonds between the sugars and phosphates in the new backbone.
• 3. Termination and Joining:
  • DNA Ligase: Hooks the ends of the old and new strands together and connects fragments.

Directionality and Strand Elongation

• Chemical Orientation: Nucleotides are bound between the $3'$ and $5'$ carbons on the deoxyribose sugar.
  • The strands run in opposite directions (anti-parallel): one strand has the $5'$ carbon/phosphate group sticking out, while the other has the hydroxyl ($OH$) group on the $3'$ carbon sticking out.
• DNA Polymerase Constraints: DNA polymerase can only add nucleotides to the $3'$ end, meaning it always builds in the $5' \rightarrow 3'$ direction.
• Leading Strand:
  • Requires only one primer.
  • DNA polymerase builds the new strand continuously as the helix opens.
• Lagging Strand:
  • Built discontinuously in pieces called Okazaki fragments.
  • Multiple primers must be added as the DNA unwinds further.
  • RNA primers are subsequently removed by DNA polymerase and replaced with DNA.
  • DNA ligase connects the Okazaki fragments to form a continuous strand.
• Error Correction: DNA polymerase performs proofreading, checking for "speed bumps or divots" (errors) during synthesis.

Gene Mutations

• Definition: Mistakes occurring during DNA duplication.
• Mutation Rates: Every gene has a characteristic mutation rate, defined as the probability of a mutation occurring in a given time period.
  • Estimated average mutation rate: Approximately $2.5 \times 10^{-8}$ mutations per nucleotide site.
  • This equates to roughly $175$ mutations per diploid genome per generation.
  • Very rare: Occurs in approximately $1$ gamete in $100,000$ to $1,000,000$.
• Base Substitution (Point Mutation):
  • A single base is swapped for another.
  • This may or may not change the resulting codon.
  • Can have large or small effects on the resulting protein.
• Frame Shift Mutations:
  • Result from insertions or deletions of nucleotides.
  • Since tRNA reads bases in groups of three (codons), adding or subtracting a base shifts the entire reading frame, changing every amino acid coded for thereafter.

Transposons and Mutagens

• Transposons ("Jumping Genes"): Pieces of DNA that can move or self-insert into different locations within the genome.
  • Examples include corn pigment variations and L1 factors.
  • Formerly thought to be rare, they are now known to be widespread, making up as much as $17\%$ of the human genome.
• Mutagens: External factors that increase the mutation rate.
  • Chemicals: Various chemical agents that interfere with DNA.
  • Ionizing Radiation: Damages DNA directly (e.g., X-rays).
  • Nonionizing Radiation: UV rays that energize cells, which in turn causes damage to the DNA structure.