DNA and the Gene: Synthesis & Repair – Study Notes

DNA and the Gene: Synthesis & Repair

What Are Genes Made Of?

Early researchers debated whether genes were composed of DNA or protein, as chromosomes contain both. Most biologists initially favored proteins due to their complexity and variability, while DNA was thought to be too simple, being made of only four types of nucleotides.

• Gene: A segment of DNA that encodes functional products, typically proteins.

• Chromosome: A structure composed of DNA and protein, carrying genetic information.

• Key Point: The chemical nature of genes was a major question in early genetics.

The Hershey–Chase Experiment

Hershey and Chase used the T2 bacteriophage to determine whether DNA or protein is the genetic material. The experiment involved infecting Escherichia coli with viruses labeled with radioactive isotopes.

• Experimental Design:

  • 32P labels DNA; 35S labels protein.

  • Labeled viruses infect E. coli cells.

  • After infection, only radioactive DNA entered the cells, not protein.

• Conclusion: Genes are composed of DNA.

Component	Radioactive Label	Location After Infection
DNA	32P	Inside cell (pellet)
Protein	35S	Outside cell (solution)

The Secondary Structure of DNA

DNA's structure is essential for its function as genetic material. Each strand has a backbone of sugar and phosphate, with nitrogenous bases projecting from the backbone.

• Primary Structure:

  1. Sugar-phosphate backbone (deoxyribonucleotides)

  2. Nitrogen-containing bases (A, T, G, C)

• Directionality:

  • 3' end: Exposed hydroxyl group on 3' carbon

  • 5' end: Exposed phosphate group on 5' carbon

Base Type	Bases	Structure
Purine	Adenine (A), Guanine (G)	Double ring
Pyrimidine	Thymine (T), Cytosine (C), Uracil (U, in RNA)	Single ring

Base Pairing: Purine + Pyrimidine: A + T or G + C

Watson and Crick Model

Watson and Crick proposed that DNA consists of two antiparallel strands forming a double helix, stabilized by complementary base pairing.

• Antiparallel Strands: Strands run in opposite directions (5' to 3' and 3' to 5').

• Double Helix: Twisted ladder structure.

• Complementary Base Pairing:

  • Adenine (A) pairs with Thymine (T) via hydrogen bonds.

  • Guanine (G) pairs with Cytosine (C) via hydrogen bonds.

DNA Polymerization and Phosphodiester Bonds

During DNA synthesis, nucleotides are joined by phosphodiester bonds between the phosphate group of the incoming nucleotide and the 3' hydroxyl group of the last nucleotide.

• Phosphodiester Bond Formation: $\text{Phosphate group (5') + Hydroxyl group (3')}$

• Directionality: DNA is synthesized in the 5' → 3' direction.

Models of DNA Replication

Three hypotheses were proposed for how parental and daughter DNA strands behave during replication:

• Semiconservative Replication: Each daughter DNA has one old (parental) and one new strand.

• Conservative Replication: Parental molecule remains intact; daughter molecule is entirely new.

• Dispersive Replication: Each daughter strand contains interspersed segments of old and new DNA.

Model	Result After 1 Generation
Semiconservative	Hybrid DNA (one old, one new strand)
Conservative	One molecule all old, one all new
Dispersive	Each strand is a mix of old and new DNA

Meselson-Stahl Experiment

Meselson and Stahl used isotopic labeling to distinguish between the replication models. E. coli was grown in "heavy" nitrogen ($^{15}$N), then transferred to "light" nitrogen ($^{14}$N), and DNA was separated by density using centrifugation.

• Experimental Setup:

  1. Grow cells in $^{15}$N medium.

  2. Transfer to $^{14}$N medium, allow one division.

  3. Isolate DNA and centrifuge to separate by density.

• Results: After one generation, DNA was of intermediate density, supporting semiconservative replication.

Generation	DNA Density
0	Heavy ($^{15}$N)
1	Intermediate (hybrid)
2	Half intermediate, half light ($^{14}$N)

A Model for DNA Synthesis

DNA synthesis is catalyzed by the enzyme DNA polymerase, which adds nucleotides only to the 3' end of a growing DNA chain. DNA synthesis always proceeds in the 5' → 3' direction.

• DNA Polymerase: Enzyme responsible for DNA synthesis; several types exist.

• Monomers: Deoxyribonucleoside triphosphates (dNTPs) provide energy for bond formation.

• Energy Source: Hydrolysis of dNTPs releases energy, making phosphodiester bond formation exergonic.

Equation: $\text{(dNMP)}_n + \text{dNTP} \rightarrow \text{(dNMP)}_{n+1} + PP_i$

Initiation of DNA Replication

Replication begins at specific sequences called origins of replication. In bacteria, there is one origin per chromosome; eukaryotes have many. Each replication bubble has two replication forks, and synthesis is bidirectional.

• Replication Bubble: Region where DNA is unwound for synthesis.

• Replication Fork: Y-shaped region where new DNA strands are synthesized.

Opening and Stabilizing the Helix

Several proteins are involved in unwinding and stabilizing the DNA double helix:

• DNA Helicase: Breaks hydrogen bonds to separate DNA strands.

• Single-strand DNA-binding proteins (SSBPs): Prevent separated strands from reannealing.

• Topoisomerase: Relieves tension by cutting and rejoining DNA.

Leading Strand Synthesis

The leading strand is synthesized continuously toward the replication fork in the 5' → 3' direction.

• DNA Polymerase: Requires a 3' end to add nucleotides; uses a sliding clamp and a grip.

• Primer: Short RNA strand (about 10 nucleotides) synthesized by primase (an RNA polymerase).

• Process: DNA polymerase adds dNTPs to the primer's 3' end.

Lagging Strand Synthesis

The lagging strand is synthesized discontinuously, away from the replication fork, in short fragments called Okazaki fragments.

• Discontinuous Replication Hypothesis:

  • Primase synthesizes new RNA primers as the fork opens.

  • DNA polymerase synthesizes short DNA fragments.

  • Fragments are joined into a continuous strand by DNA ligase.

The Replisome: A Molecular Machine

The replisome is a large complex containing all the enzymes required for DNA synthesis at the replication fork.

• Components: DNA polymerase, primase, helicase, SSBPs, topoisomerase, DNA ligase, sliding clamp.

• Function: Coordinates leading and lagging strand synthesis.

Summary Table: Enzymes Involved in DNA Replication

Enzyme	Function
DNA Polymerase	Synthesizes new DNA strands
Primase	Synthesizes RNA primers
DNA Helicase	Unwinds DNA helix
SSBPs	Stabilize single-stranded DNA
Topoisomerase	Relieves supercoiling/tension
DNA Ligase	Joins Okazaki fragments

Key Learning Objectives

• Describe experiments that demonstrated DNA is the hereditary material.

• Explain the semiconservative model of DNA replication and supporting evidence.

• List and describe the roles of enzymes involved in leading and lagging strand replication.

Example: Okazaki Fragment Synthesis

• Primase synthesizes an RNA primer.

• DNA polymerase III extends the fragment.

• DNA polymerase I replaces RNA primer with DNA.

• DNA ligase seals the gap between fragments.

Additional info: These notes expand on the provided slides with definitions, explanations, and tables for clarity and completeness, suitable for Genetics college students.