DNA Structure and Replication

DNA Structure

  • Definition of DNA: DNA stands for deoxyribonucleic acid.

    • Nature of DNA: This chemical substance is found in the nucleus of all cells in all living organisms.

    • Function: DNA controls all the chemical changes that occur within cells, influencing:

    • The type of cell that is formed (e.g., muscle, blood, nerve cells).

    • The kind of organism that is produced (e.g., buttercup, giraffe, herring, human).

DNA Overview

  • Composition of DNA:

    • DNA is a large biomolecule made up of long chains of subunits called nucleotides.

    • Each nucleotide is comprised of:

    • A sugar called deoxyribose.

    • A phosphate group denoted as -PO4.

    • An organic base.

Deoxyribose and Ribose

  • Comparison: Ribose is a sugar similar to glucose, with five carbon atoms in its molecule.

  • Deoxyribose: Deoxyribose resembles ribose but lacks one oxygen atom compared to ribose.

Nitrogenous Bases

  • Types of Nitrogenous Bases:

    • Purines: Contain two carbon-nitrogen rings with amino functional groups.

    • Pyrimidines: Contain a single carbon-nitrogen ring with amino functional groups.

Major Events in DNA Research

  • 1869: Johann Miescher discovers an acidic substance in the nucleus containing nitrogen and phosphorus (DNA).

  • 1928: Frederick Griffith identifies a “transforming factor” in bacteria.

  • 1944: Avery determines that DNA is the transforming factor, though it is not widely accepted initially.

  • 1952:

    • Hershey-Chase Experiment: Demonstrates that DNA is the hereditary material and not protein (widely accepted).

  • 1952: Chargaff conducts experiments establishing the base pairings of DNA, revealing that A & T were more common than C & G and that the amounts of A/T and C/G are equal.

  • 1952: Rosalind Franklin utilizes X-ray diffraction to obtain the first clear picture of DNA.

  • 1953: James Watson and Francis Crick develop the molecular model of DNA, which is the double helix.

Structure of DNA

  • Description: DNA structure resembles a twisted ladder, known as a double helix.

    • Backbone Structure: Made up of deoxyribose and phosphate groups of each nucleotide.

    • Steps of the Ladder: Composed of nitrogenous bases (A, G, C, T).

    • Base Pairing: Any sequence of bases is possible, held together by hydrogen bonds.

  • Historical Context: Other models of DNA were proposed, but only the double helix model met both Chargaff’s data and Franklin’s X-ray observations.

Incorrect DNA Structure Models

  • Triple Helix Model: Proposed by Linus Pauling just prior to Watson and Crick's model.

    • Flaws: Incorporates phosphate groups on the inside, with nucleotides pointing outward, which results in:

    • Negatively charged phosphates repelling each other.

    • Weak interactions that do not adequately hold the structure together.

Directionality of DNA

  • Strand Directionality:

    • Phosphate on the 5’ carbon of one nucleotide bonds to the 3’ carbon of the next nucleotide.

    • This establishes a direction for each DNA strand, starting from 5’ end to 3’ end.

    • The strands in the double helix are antiparallel:

    • One strand runs 5’ to 3’ while the complementary strand runs 3’ to 5’.

Anti-parallel Strands of DNA

  • Bonding in the DNA Backbone:

    • Nucleotides are covalently bonded via phosphodiester bonds between the 3’ and 5’ carbons, giving the DNA molecule a defined direction.

    • Each complementary strand runs in the opposite direction:

    • One strand: 5’ to 3’.

    • Complementary strand: 3’ to 5’.

Chargaff’s Rule

  • Chargaff's Analysis: Analyzed DNA across various organisms and found:

    • Adenine (A) always equals Thymine (T).

    • Guanine (G) always equals Cytosine (C).

  • Significance: Establishes that a purine always bonds to a pyrimidine.

Chromosome Structure in Prokaryotes

  • Characteristics:

    • Example: E. coli bacterium.

    • Structure: The DNA molecule is a single, double-stranded circular loop.

    • Replication process is generally similar across different DNA structures.

  • Size: Contains approximately 5 million base pairs and 3,000 genes.

DNA Replication: General Overview

  • Purpose: Cells must replicate DNA before division to ensure each daughter cell inherits a complete genetic copy.

  • Models of Replication:

    • Semiconservative: Each strand serves as a template.

    • Conservative: Entire DNA molecule acts as a template.

    • Dispersive: Existing strands are broken and mixed with new segments.

Meselson and Stahl Experiment

  • Experiment Outline: Bacteria cultured in a medium containing heavy nitrogen (15N) were transferred to a medium with lighter nitrogen (14N).

  • Observations:

    • After the first replication, a mixture of heavy and light DNA was seen (indicative of semi-conservative replication).

    • Following the second replication, lighter DNA predominated.

Semi-Conservative Model

  • Mechanism: During DNA replication,

    • Each strand serves as a template, resulting in new strands comprised of half parental template and half new DNA.

Steps in DNA Replication

  1. Coordination by Enzymes: A variety of enzymes work together at the replication fork.

    • Similar Processes: Occurs in both eukaryotes and prokaryotes.

  2. Initial Unwinding:

    • DNA Gyrase: Unwinds supercoiled DNA.

    • Helicase Enzyme: Separates hydrogen bonds between base pairs.

    • Single-Stranded Binding Proteins: Stabilize unwound DNA, preventing reformation of hydrogen bonds.

  3. RNA Primer Addition:

    • Primase: Adds a small section of RNA known as the RNA primer at the 3' end of the template DNA.

    • In eukaryotes, primase operates with DNA polymerase α to synthesize DNA-RNA primers.

  4. Building New Strands:

    • DNA Polymerase III: Synthesizes new daughter strands by adding complementary bases to the parent strands.

    • In eukaryotes, DNA polymerase δ interacts with Proliferating Cell Nuclear Antigen (PCNA), acting as a sliding clamp.

  5. Completing the Replication:

    • DNA Polymerase I: Removes RNA primers and fills in the gaps with DNA in prokaryotes. Similarly completed by DNA polymerase δ in eukaryotes.

    • DNA Ligase: Joins fragments of DNA into a cohesive strand that is then wound tightly into structure.

Okazaki Fragments

  • Observation: Both DNA strands must be replicated in both directions. Since DNA polymerases can only operate in a 5’ to 3’ direction, this leads to:

    • Leading Strand: Replicated continuously in one long pass.

    • Lagging Strand: Replicated in short segments known as Okazaki fragments.

Overall Summary of Replication Steps**

  1. Helicase: Separates the DNA strands.

  2. Single-Stranded Binding Proteins (SSB): Prevent re-annealing.

  3. Primase: Synthesizes RNA primers.

  4. DNA Polymerase I: Removes RNA primers and fills in gaps.

  5. DNA Ligase: Joins DNA fragments together.

  6. Leading & Lagging Strands: Results in one leading strand and one lagging strand due to directionality constraints.