DNA Replication

Learning Objectives and Genetic Material Characteristics

  • Core Learning Objectives:

    • To comprehend the significance and mechanism of semi-conservative replication.
    • To define and understand the specific functions of DNA polymerases, helicase, and DNA ligase.
    • To provide a detailed description of the key stages involved in the DNA replication process.

  • Required Reading Assignments:

    • T. Brown, 2011: Genomes 3. Chapter 10 (pages 187–200, 205–208) and Chapter 11 (pages 221-223).
    • Klug, 2016: Concepts of Genetics. Chapter 11 (pages 295-318).

  • Crucial Characteristics of Genetic Material:

    • For any molecule to function as the genetic material, it must satisfy four criteria:
      1. Storage of Information: Must hold the blueprints for the organism.
      2. Replication: Must be capable of being copied accurately during the cell cycle.
      3. Expression of Information: Must be able to direct the synthesis of proteins (the Central Dogma).
      4. Variation by Mutation: Must allow for changes that provide the raw material for evolution.

The Central Dogma and Genome Scale

  • The Central Dogma Framework:

    • The foundational concept states: "DNA makes RNA, which makes proteins."
    • Replication: DNA $\rightarrow$ DNA (facilitated by DNA Polymerase).
    • Transcription: DNA $\rightarrow$ RNA (facilitated by RNA Polymerase).
    • Translation: RNA $\rightarrow$ Protein (facilitated by Ribosomes).

  • The Scale of the Human Genome:

    • The human genome consists of approximately 3.23.2 billion base pairs (3,200,000,000 bp3,200,000,000\,bp).
    • Fidelity Requirements: This massive amount of data must be replicated faithfully. Even a minor error rate of 11 in 1,000,0001,000,000 base pairs would result in 3,2003,200 errors per replication cycle, which is considered excessively high. While replication is not entirely error-free, it is remarkably accurate.

Theoretical Models of DNA Replication

  • In the mid-1950s, three competing models were proposed for how DNA replicates:
    1. Semi-conservative Model: Results in two hybrid molecules, where each double helix consists of one "old" (parental) strand and one "new" (synthesized) strand. This was the model proposed by Watson and Crick.
    2. Conservative Model: The parental double helix is conserved as a whole, and an entirely new double helix molecule is produced.
    3. Dispersive Model: Results in hybrid molecules where every individual strand is a mixture or mosaic of old and new DNA segments.

The Meselson-Stahl Experiment (1958)

  • Isotopic Background:

    • The experiment utilized two isotopes of nitrogen: the naturally occurring "light" 14N{^{14}N} and the "heavy" isotope 15N{^{15}N}, which contains one additional neutron.
    • DNA contains nitrogen in its nitrogenous bases (purines and pyrimidines).
    • Density gradient centrifugation (using Cesium Chloride, CsClCsCl) allows for the separation of DNA molecules based on their weight; 15N{^{15}N}-DNA sediments lower (is heavier) than 14N{^{14}N}-DNA.

  • Experimental Procedure:

    1. E. coli bacteria were grown for many generations in a medium where 15NH4Cl{^{15}NH_4Cl} (ammonium chloride) was the sole nitrogen source. This ensured all DNA was uniformly labeled with "heavy" nitrogen.
    2. Observation at Generation 0: After centrifugation, a single heavy band was observed (100% 15N100\%\, {^{15}N}).
    3. The cells were then transferred to a medium containing only "light" 14N{^{14}N}.
    4. Observation at Generation 1: After one replication cycle (approx. 20 minutes20\,\text{minutes}), centrifugation showed a single band of intermediate density (100% 15N/14N100\%\, {^{15}N}/{^{14}N} hybrids). This immediately ruled out the conservative model.
    5. Observation at Generation 2: After two cycles (40 minutes40\,\text{minutes}), two bands appeared: one at intermediate density and one at light density (50% 15N/14N50\%\, {^{15}N}/{^{14}N} and 50% 14N/14N50\%\, {^{14}N}/{^{14}N}). This ruled out the dispersive model.
    6. Successive Generations: In Generation 3 (60 minutes60\,\text{minutes}), the proportions were 25% hybrid25\%\,\text{hybrid} and 75% light75\%\,\text{light}. In Generation 4 (80 minutes80\,\text{minutes}), the result was 12% hybrid12\%\,\text{hybrid} and 88% light88\%\,\text{light}.

  • Conclusion: DNA replication is semi-conservative, providing each daughter molecule with one parental strand and one newly synthesized strand.

Structural Requirements and Enzymology

  • Polynucleotide Directionality:

    • Synthesis always occurs in the 5′→3′5' \rightarrow 3' direction.
    • The template strand is read in the 3′→5′3' \rightarrow 5' direction.
    • Nucleotides are attached to the sugar via a β\beta-N-glycosidic bond at the 1′C1'C and linked via phosphate groups attached to the 5′C5'C and 3′C3'C.

  • Essential Components for Synthesis:

    1. Template DNA: To guide the sequence.
    2. dNTPs (Deoxyribonucleotide triphosphates): dATP, dGTP, dTTP, and dCTP serve as substrates.
    3. DNA Polymerase: Requires Magnesium ions (Mg2+Mg^{2+}) as a cofactor.
    4. RNA Primer: Provides a free 3′−OH3'-OH group for the polymerase to begin adding nucleotides.

  • Key Enzymes and Their Roles:

    • Topoisomerase: Unwinds the DNA double helix and "relaxes" the backbone to prevent supercoiling (acts as a swivel).
    • DNA Helicase: Separates the two polynucleotide strands by breaking the hydrogen bonds between bases.
    • Single-stranded Binding Proteins (SSBP): Stabilize the separated strands and prevent them from reannealing.
    • Primase: A specialized RNA polymerase that synthesizes short RNA primers.
    • DNA Polymerase III: The primary enzyme for adding nucleotides to the 3′3' end of the growing strand in prokaryotes.
    • DNA Polymerase I: In prokaryotes, it removes the RNA primer and fills the resulting gaps with DNA nucleotides.
    • DNA Ligase: Seals the "nicks" in the sugar-phosphate backbone by forming covalent phosphodiester linkages between the 3′-hydroxyl3'\text{-hydroxyl} and 5′-phosphate5'\text{-phosphate} groups.

The Mechanism of Replication

  • Origins of Replication:

    • Prokaryotes: Typically have a single origin (autonomously replicating sequence or ARS), usually rich in AT base pairs.
    • Eukaryotes: Have multiple origins termed "replicons," which appear as "replication bubbles" under an electron microscope. Each bubble has two replication forks. In G1 phase, these are recognized by the six-protein Origin Recognition Complex (ORC).

  • Stages of Replication:

    1. Initiation: Helicase unwinds the DNA at the origin. SSBPs stabilize the strands. Primase creates an RNA primer. DNA polymerase attaches to the 3′3' end of the primer.
    2. Elongation:
      • Leading Strand: Synthesis is continuous as the polymerase moves toward the replication fork in the 5′→3′5' \rightarrow 3' direction.
      • Lagging Strand: Synthesis is discontinuous and moves away from the fork. It produces short segments (approx. 1000−2000 bp1000-2000\,bp in prokaryotes) known as Okazaki fragments. Each fragment requires its own RNA primer.
    3. Termination: RNA primers are replaced by DNA, and fragments are joined by ligase.

Fidelity, Errors, and Structural Limitations

  • Proofreading Activity:

    • DNA Polymerase has a built-in proofreading mechanism to correct errors (initial error rate: 11 in 10610^6).
    • It possesses 3′→5′3' \rightarrow 5' exonuclease activity, identifying an incorrect base, cleaving the phosphodiester bond to remove it, and then resuming synthesis in the 5′→3′5' \rightarrow 3' direction.

  • The End-Replication Problem:

    • DNA polymerases can only synthesize in the 5′→3′5' \rightarrow 3' direction and cannot initiate synthesis without a primer.
    • On linear eukaryotic chromosomes, there is no way to replace the RNA primer at the extreme 3′3' end of the lagging strand. This causes chromosomes to shorten with every round of replication.
    • Solution: Telomeres (repetitive DNA sequences at chromosome ends) and the enzyme Telomerase, which maintains these ends. Telomerase is active during embryonic development but generally inactive in adult somatic cells.

Packaging of DNA

  • Human DNA is extensively organized to fit inside the cell nucleus:
    • Chromatin: The complex formed by DNA and proteins.
    • Histones: DNA-binding proteins that facilitate the compacting and folding of DNA.
    • Nucleosome: The fundamental unit of chromatin, consisting of DNA coiled around a cluster of histone proteins.

Questions & Discussion

  • Question: Do we have nitrogen in DNA?
  • Response: Yes, the nitrogenous bases (Purines and Pyrimidines) are the nitrogen-containing components of nucleotides. Purines are double-ringed bases, while Pyrimidines are single-ringed.
  • Question: What happens to 15N{^{15}N} labeled cells when switched to 14N{^{14}N} medium?
  • Response: The newly synthesized strands will incorporate the light nitrogen (14N{^{14}N}), allowing researchers to track the distribution of the original heavy parental material through successive generations.

Homework and Logistics

  • Task: Create a single-page A4 drawing explaining the Meselson-Stahl (1958) experiment. Must include both words and drawings clearly stating what happens at each step.
  • Submission: Include name, student number, and group number. Take a photo, paste it into a Word document, and upload via Turnitin.
  • Deadline: 7th May 2020.
  • Next Topic: Gene expression: Transcription and The Genetic Code. Reading assigned from T.A. Brown, 2011, Chapters 4 and 7.