IB Biology D1.1 DNA Replication Study Guide

Fundamental Questions and Importance of DNA Replication

Core Concepts: DNA replication addresses two primary questions in biology:
- How is new DNA produced?
- How has knowledge of DNA replication enabled applications in biotechnology?
Biological Necessity of Replication (D1.1.1): DNA replication is required for:
- Reproduction: Passing genetic information to offspring.
- Growth: Increasing the number of cells in an organism.
- Tissue Replacement: Replacing damaged or dead cells in multicellular organisms.
Timing: DNA is copied during the S phase (synthesis phase) of interphase before a cell divides via Mitosis (cell reproduction) or Meiosis (gamete production).

Structural Fundamentals and Bonding in DNA

The DNA Backbone: Composed of sugar and phosphate groups.
- Bonding: Sugar and phosphate are covalently bonded via phosphodiester bonds.
Nitrogenous Bases: Lie between the sugar-phosphate backbones.
Hydrogen Bonding: Complementary base pairs are held together by H-bonds:
- Adenine ( $A$ ) to Thymine ( $T$ ): $2$ H-bonds.
- Guanine ( $G$ ) to Cytosine ( $C$ ): $3$ H-bonds.
Antiparallel Strands: Defined by the $3'$ and $5'$ terminals of the nucleotide strands.

Characteristics and Models of Replication

Replication Criteria:
- Fidelity: The process requires an exquisite degree of accuracy. Uncorrected errors can permanently affect gene function or eliminate it, and such changes are inheritable.
- Speed: DNA molecules can contain millions of bases. The machinery (proteins and enzymes) must synthesize DNA with extraordinary speed.
The Semiconservative Model (D1.1.2):
- Definition: Both daughter molecules contain one strand of the parent molecule (the template) and one strand of newly synthesized DNA.
- Accuracy: This process allows for a high degree of accuracy by using the parent strand as a template for complementary base pairing.
Inaccurate Models:
- Conservative Model: Two parent strands remain together; a completely new daughter molecule is made.
- Dispersive Model: Daughter molecules contain a mix of parent and new DNA throughout both strands.
The Meselson-Stahl Experiment (1958):
- Procedure: Bacteria were cultured in a medium with $^{15}N$ (heavy isotope of Nitrogen) and then transferred to a medium with $^{14}N$ (lighter isotope).
- Results after 1st replication: DNA sample centrifuged showed a band of intermediate density.
- Results after 2nd replication: DNA sample showed both intermediate and light density bands.
- Conclusion: This experiment proved the semiconservative model of replication.

General Mechanism of DNA Replication (D1.1.3)

Rules of Replication:
- Replication is semiconservative.
- Replication begins at an origin and usually proceeds bidirectionally.
- Synthesis proceeds in a $5' \rightarrow 3'$ direction and is semidiscontinuous.
Enzymes and Proteins:
- Helicase: Unwinds the DNA double helix and breaks the hydrogen bonds between the two strands, creating the replication fork.
- DNA Gyrase (Topoisomerase II): Unties knots or supercoils that form ahead of the replication fork.
- Single-Stranded Binding Proteins (SSB): Stabilize the unwound DNA and prevent the strands from immediately rewinding.
- DNA Polymerase III: Known as the 'replicase' enzyme; it adds new complementary bases to the daughter strand.
- DNA Polymerase I: A repair enzyme involved in removing RNA primers, filling in gaps, and correcting typos.
- DNA Ligase: Seals 'nicks' in the sugar-phosphate backbone by joining fragments.
- RNA Polymerase / Primase: Creates RNA primers to initiate synthesis for DNA Polymerase III.
- RNase H: Removes RNA primers once they have completed their function.

Advanced Aspects of Directionality and Synthesis (AHL - D1.1.6, D1.1.7, D1.1.8)

Directionality of DNA Polymerases:
- DNA polymerases add the $5'$ end of a DNA nucleotide to the $3'$ end of an existing strand.
- Strands can only grow in the $5' \rightarrow 3'$ direction.
- Energy for Bonding: Nucleotides arrive as nucleoside triphosphates (ATP, GTP, TTP, CTP). Bonding energy is provided by the cleavage of the extra phosphate groups (P-P-P).
Leading vs. Lagging Strands:
- Leading Strand: Synthesis is continuous. It proceeds in the same direction as the replication fork movement. Only one RNA primer is required.
- Lagging Strand: Synthesis is discontinuous. It proceeds away from the replication fork. It requires repeated RNA primers and is synthesized in short segments called Okazaki fragments.
The Prokaryotic System (E. coli):
- DNA Primase: Synthesizes a short RNA primer to provide a $3'$ end for Polymerase III.
- DNA Polymerase III: Main builder ( $1000$ bases per second).
- DNA Polymerase I: Removes RNA primers and replaces them with DNA nucleotides ( $20$ bases per second).
- DNA Ligase: Functions as a "spot welder" to join Okazaki fragments together.

Accuracy, Proofreading, and Maintenance (AHL - D1.1.9)

Speed and Fidelity Statistics:
- Human cell: Copies $6 \times 10^9$ bases and divides in a few hours.
- E. coli: Takes less than $1$ hour to copy $5 \times 10^6$ base pairs in its single chromosome.
- Error Rates (Replication Steps):
  - $5' \rightarrow 3'$ polymerization: $1 \times 10^5$
  - $3' \rightarrow 5'$ proofreading: $1 \times 10^2$
  - Strand-directed mismatch repair: $1 \times 10^2$
  - Total error rate: approximately $1 \times 10^9$ ( $1$ error per $1$ billion bases).
Proofreading Mechanisms:
- DNA Polymerase III: If a mismatched base is detected at the $3'$ terminal, it is removed and replaced with the correct nucleotide.
- Mismatch Repair: A protein complex binds the mispaired base, a second complex cuts the DNA, enzymes excise the patch, DNA Polymerase replaces the section, and Ligase seals it.
- Distinguishing Strands: Enzymes use methyl groups ( $CH_3$ ) on the old template strand to identify which strand is correct and which is the new (potentially erroneous) strand.
Environmental Damage: Errors can arise from carcinogens like X-rays, UV light (causing thymine dimers), and cigarette smoke.
Telomeres: Highly repetitive DNA sequences at the ends of eukaryotic chromosomes. They protect genes from being deleted during replication, as the lagging strand leaves a gap at the end. Shortening of telomeres is linked to aging.
Tandem Repeats: Short sequences (2-5 base pairs) of non-coding DNA repeated numerous times in a head-to-tail manner (e.g., GATA).

Biotechnology: PCR and Gel Electrophoresis (D1.1.4, D1.1.5)

Polymerase Chain Reaction (PCR):
- Purpose: Amplifying small quantities of specific DNA target sequences (e.g., from crime scenes).
- Process in a Thermal Cycler:
  1. Denaturation: Heat is used to separate DNA into two strands.
  2. Annealing: DNA primers attach to the ends of the target sequence.
  3. Elongation: Taq polymerase (heat-tolerant) copies the strands.
- Yield: Each cycle doubles the DNA. $30$ cycles yield $2^{30} \approx 1,073,741,826$ copies.
Gel Electrophoresis:
- Mechanism: Separates DNA fragments based on size using an electrical field.
- Principles: DNA is negatively charged and moves toward the positive electrode. Small pieces travel faster and farther through the agarose gel (derived from algae) than large pieces.
- Visualization: Fragments are stained with Ethidium bromide, which fluoresces under UV light.
Applications of DNA Profiling:
- Forensics: Comparing crime scene samples (blood, semen, hair) with suspect DNA. Notable case: 1987 rape case of Tommie Lee Andrews (first successful use); also used in the OJ Simpson trial.
- Paternity Testing: Comparing banding patterns of mother, child, and potential fathers.
- Medical Diagnostics: Comparing normal alleles to disease-causing alleles (e.g., Huntington's disease).
- Evolutionary Relationships: Comparing DNA between different species (e.g., turtle, snake, rat, squirrel, fruit fly).
Nature Of Science (NOS): Reliability in DNA profiling is enhanced by increasing the number of genetic markers used, which reduces the probability of a false match.