DNA Structure and Replication

Introduction to Nucleic Acids and Genetic Material

  • Nucleic Acids as Major Biological Molecules:

    • Nucleic acids represent a major category of biologically significant molecules.

    • There are two primary types of nucleic acids:

      • Deoxyribonucleic Acid (DNA): The molecule that carries genetic information.

      • Ribonucleic Acid (RNA): Involved in various roles, primarily protein synthesis and regulation.

  • Griffith’s Transformation Experiment (1928):

    • Subject: Streptococcus pneumoniae.

    • Observation: Griffith observed the phenomenon of "Transformation."

    • Experimental Setup:

      1. Live Bacterial Strain A (R Strain): Non-virulent; does not kill the mouse.

      2. Dead Bacterial Strain B (Heat-killed S Strain): Virulent but rendered inactive by heat; does not kill the mouse.

      3. Combination: Live Strain A + Dead Strain B resulted in a Live Bacterial Strain A with a new genotype and phenotype (virulence).

    • The Transformation Factor: The heat-killed virulent S strain transferred "something" to the live R strain, leading to virulence. This question of "what part of the dead S strain was transferred?" remained the central mystery.

  • Avery, MacLeod, & McCarty’s Clarification (1944):

    • This experiment was a variation of Griffith's, designed to identify the specific molecule responsible for transformation.

    • Process: They used an extract from the S strain and systematically destroyed single molecular components to see which destruction prevented the recovery of live S strain (and thus let the mouse live).

    • Results:

      • Polysaccharides destroyed: Mouse dies; Live S strain recovered.

      • Lipids destroyed: Mouse dies; Live S strain recovered.

      • RNA destroyed: Mouse dies; Live S strain recovered.

      • Protein destroyed: Mouse dies; Live S strain recovered.

      • DNA destroyed: Mouse lives; No live S strain recovered.

    • Conclusion: DNA is the transforming principle.

  • Hershey & Chase Experiment (1952) - The Blender Experiment:

    • Subject: T2 Bacteriophage (a virus that infects bacteria) and E. coli.

    • Hypothesis: Determine if protein or DNA is the genetic material by labeling each.

    • Labeling Strategy:

      1. DNA Labeling: Used radioactive Phosphorus (32P^{32}P). Phosphorus is found in DNA but not in protein.

      2. Protein Labeling: Used radioactive Sulfur (35S^{35}S). Sulfur is found in protein (amino acids like cysteine/methionine) but not in DNA.

    • Methodology:

      • Infect bacteria with labeled phages.

      • Agitate in a blender to detach "phage ghosts" (empty protein shells) from the bacterial surface.

      • Centrifuge to separate the heavy bacteria (pellet) from the lighter phage ghosts (supernatant).

    • Results:

      • When protein was labeled (35S^{35}S), most radioactivity was recovered in the phage ghosts (outside the bacteria).

      • When DNA was labeled (32P^{32}P), most radioactivity was recovered inside the bacteria.

    • Conclusion: DNA, not protein, is the genetic molecule injected into bacteria to direct the production of new viruses.

Chemical Structure of DNA

  • The Nucleotide Unit:

    • DNA is composed of repeating units called deoxynucleotides.

    • Components of a Deoxyribonucleotide:

      • Phosphate Group: Attached to the 5′5' carbon of the sugar.

      • Deoxyribose Sugar: A five-carbon (pentose) sugar. Carbon numbering follows: 1′1' (attached to base), 2′2' (has H in DNA, OH in RNA), 3′3' (attached to the next phosphate), 4′4', and 5′5' (attached to the phosphate of the current nucleotide).

      • Nitrogenous Base: Attached to the 1′1' carbon.

  • Categories of Nitrogenous Bases:

    • Purines (Two-ring structure):

      • Adenine (A)

      • Guanine (G)

    • Pyrimidines (One-ring structure):

      • Cytosine (C)

      • Thymine (T) (Note: In RNA, Uracil (U) replaces Thymine).

  • Phosphodiester Bonds and Strand Polarity:

    • Adjacent nucleotides are linked by phosphodiester bonds.

    • The bond forms through a condensation reaction between the 5′5' phosphate of one nucleotide and the 3′3' hydroxyl (OHOH) group of the preceding nucleotide.

    • Polarity: DNA strands have a distinct directionality, referred to as 5′→3′5' \rightarrow 3'.

      • The 5′5' end terminates in a phosphate group.

      • The 3′3' end terminates in a hydroxyl group.

  • Chargaff’s Rules:

    • Based on chemical analysis of DNA across species:

    1. Total Purines = Total Pyrimidines: A+G=T+CA + G = T + C.

    2. Specific Ratios: The amount of Adenine equals Thymine (A=TA = T) and the amount of Guanine equals Cytosine (G=CG = C).

The DNA Double Helix

  • Physical Properties:

    • DNA is a double helix composed of two nucleotide chains.

    • The structure is right-handed.

    • The two chains are anti-parallel, meaning they run in opposite directions (one 5′→3′5' \rightarrow 3', the other 3′→5′3' \rightarrow 5').

  • Stabilizing Forces:

    • Hydrogen Bonds: The two strands are held together by hydrogen bonds between complementary nitrogenous bases.

    • Base Pairing Specificity:

      • Guanine and Cytosine (G:CG:C): Held by three hydrogen bonds (stronger bond).

      • Adenine and Thymine (A:TA:T): Held by two hydrogen bonds (weaker bond).

DNA Replication in Prokaryotes

  • General Characteristics:

    • Template: Occurs in a single, circular DNA molecule.

    • Origin: There is one specific origin of replication.

    • Directionality: Replication is bi-directional and synthesis always proceeds in the 5′→3′5' \rightarrow 3' direction.

    • Method: Semi-conservative replication, where each daughter DNA molecule consists of one original (parental) strand and one newly synthesized strand.

  • The Replication Fork and Unwinding:

    • DNA Helicase: Enzyne that separates the two DNA strands by breaking hydrogen bonds.

    • Single-Strand Binding Proteins (SSB): Bind to the exposed single strands to prevent them from re-annealing (snapping back together).

    • DNA Gyrase (Topoisomerase): Relieves the torsional strain and positive supercoiling caused by the unwinding of the helix.

  • The Process of Synthesis:

    1. Initiation: DNA polymerase requires a pre-existing 3′3' end to add nucleotides. Primase (part of a primosome complex with 6 other proteins) synthesizes a short RNA primer (10–20 bases) complementary to the DNA template.

    2. Elongation:

      • DNA Polymerase III: The major replication enzyme. It adds nucleotides to the 3′3' end of the primer or growing strand. It acts as a dimer (the Pol III holoenzyme) to service both strands via strand looping.

      • Leading Strand: DNA is synthesized continuously in the same direction as the replication fork movement (5′→3′5' \rightarrow 3').

      • Lagging Strand: DNA is synthesized discontinuously in the opposite direction of the fork, resulting in short segments called Okazaki fragments.

    3. Primer Removal and Gap Filling:

      • DNA Polymerase I: Uses its 5′→3′5' \rightarrow 3' exonuclease activity to remove the RNA primers and its polymerase activity to fill the resulting gaps with DNA nucleotides. It also performs proofreading via 3′→5′3' \rightarrow 5' exonuclease activity.

    4. Ligation:

      • DNA Ligase: Seals the "nicks" (breaks in the phosphodiester backbone) between adjacent Okazaki fragments. It requires energy in the form of ATPATP or NADNAD.

  • The Replisome:

    • A complex molecular machine consisting of the Pol III holoenzyme, DnaB helicase, Primase, and the β\beta clamp (a protein that helps stabilize Pol III on the DNA).

Prokaryotic DNA Polymerases

  • Prokaryotes utilize 5 different DNA polymerases:

    • Pol I Roles:

      1. 5′→3′5' \rightarrow 3' polymerase activity.

      2. 3′→5′3' \rightarrow 5' exonuclease activity (Proofreading).

      3. 5′→3′5' \rightarrow 3' exonuclease activity (Removal of damaged DNA and RNA primers).

    • Pol III Roles:

      1. 5′→3′5' \rightarrow 3' polymerase activity.

      2. 3′→5′3' \rightarrow 5' exonuclease activity.

      3. Major enzyme for elongation during replication.

Specialized Replication Models

  • Rolling Circle Replication:

    • Common in plasmids and some viruses.

    • A nuclease cuts one strand, creating a free 3′−OH3'-OH and a 5′−P5'-P end.

    • Nucleotides are added to the 3′−OH3'-OH end, displacing the 5′5' end.

    • As the circle rolls, the displaced strand is used as a template for discontinuous (lagging strand) synthesis.

DNA Replication in Eukaryotes

  • Differences from Prokaryotes:

    • Complexity: Eukaryotic replication has a longer duration.

    • Chromosomes: Linear chromosomes rather than circular.

    • Origins: Multiple origins of replication per chromosome to handle the larger volume of DNA.

    • Polymerases: Uses 5 primary DNA polymerases: α,β,γ,δ,ϵ\alpha, \beta, \gamma, \delta, \epsilon.

  • The Telomere Problem:

    • Linear chromosomes face a problem at the ends (telomeres).

    • When the final RNA primer on the lagging strand is removed, there is no upstream 3′−OH3'-OH for DNA polymerase to fill the gap.

    • If left unaddressed, the chromosome would shorten with every round of replication, eventually losing coding regions.

  • Telomere Replication and Telomerase:

    • Telomeres: Consist of repeating non-coding sequences (e.g., TTGGGGTTGGGG in some organisms).

    • Telomerase: An enzyme carrying its own RNA template complementary to the repeating sequence.

    • Mechanism:

      1. Annealing: Telomerase binds to the 3′3' overhang of the parental DNA strand.

      2. Elongation: It uses its RNA template to extend the 3′3' end of the parental strand.

      3. Translocation: The enzyme moves down to repeat the extension.

      4. Lagging Strand Completion: Once the parental strand is sufficiently long, Primase can lay down a new RNA primer, and DNA polymerase can fill in the complementary lagging strand gap.

      5. Final Seal: The primer is removed and the gap is sealed by DNA ligase.