DNA and the Gene: Synthesis and Repair – Study Notes

DNA and the Gene: Synthesis and Repair

Introduction to Genes and Genetic Material

Understanding the molecular nature of genes and their replication was a major milestone in biology. Early experiments established the foundation of classical genetics, but the chemical identity of genes and the mechanisms of their copying and repair were not initially clear. This chapter explores the discoveries that revealed DNA as the genetic material, the process of DNA replication, and the mechanisms for repairing DNA damage.

What Are Genes Made Of?

The Chemical Nature of Genes

• Chromosomes are composed of DNA and proteins.

• Historically, proteins were thought to be the genetic material due to their complexity and variability, while DNA was considered too simple (only four nucleotides).

The Hershey–Chase Experiment

The Hershey–Chase experiment provided definitive evidence that DNA, not protein, is the hereditary material.

• T2 bacteriophage infects Escherichia coli by injecting its genetic material into the cell, leaving the protein coat outside.

• Radioactive isotopes were used to label DNA (phosphorus) and protein (sulfur).

• Only radioactive DNA entered the bacterial cells, demonstrating that genes are composed of DNA.

The Structure of DNA

DNA as a Double Helix

• DNA is a double-stranded polymer of deoxyribonucleotides.

• Each nucleotide consists of a deoxyribose sugar, a phosphate group, and a nitrogenous base.

• Nucleotides are linked by phosphodiester bonds between the 3′ hydroxyl of one sugar and the 5′ phosphate of the next.

• The backbone is formed by sugar and phosphate groups; bases project from the backbone.

Directionality and Base Pairing

• Each DNA strand has polarity: a 3′ end (hydroxyl group) and a 5′ end (phosphate group).

• Strands are antiparallel and twist into a double helix.

• Complementary base pairing: Guanine (G) pairs with Cytosine (C); Adenine (A) pairs with Thymine (T).

• The double helix is stabilized by hydrogen bonds between bases and interactions between stacked bases.

Testing Early Hypotheses About DNA Synthesis

Models of DNA Replication

• Semiconservative replication: Each daughter DNA has one old and one new strand.

• Conservative replication: One daughter has both old strands; the other has both new strands.

• Dispersive replication: Each daughter has interspersed old and new DNA segments.

The Meselson–Stahl Experiment

• Used isotopes of nitrogen to distinguish old and new DNA strands in E. coli.

• Results supported the semiconservative model of DNA replication.

A Model for DNA Synthesis

DNA Polymerase and Directionality

• DNA polymerase catalyzes DNA synthesis, adding nucleotides only to the 3′ end of a growing chain (5′ → 3′ direction).

• Monomers are deoxyribonucleoside triphosphates (dNTPs), which provide energy for bond formation.

Origin of Replication and Replication Forks

• Replication begins at specific sequences called origins of replication.

• Bacteria have one origin; eukaryotes have many per chromosome.

• Replication bubbles form, each with two replication forks moving bidirectionally.

Opening and Stabilizing the Helix

• DNA helicase separates the DNA strands by breaking hydrogen bonds.

• Single-strand DNA-binding proteins (SSBPs) prevent re-annealing.

• Topoisomerase relieves tension caused by unwinding.

Synthesis of Leading and Lagging Strands

Leading Strand Synthesis

• DNA polymerase requires a primer (short RNA strand) to begin synthesis.

• Primase synthesizes the RNA primer.

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

Lagging Strand Synthesis and Okazaki Fragments

• The lagging strand is synthesized discontinuously, away from the replication fork.

• Primase synthesizes new primers as the fork opens.

• DNA polymerase synthesizes short DNA fragments (Okazaki fragments), which are later joined.

• DNA polymerase removes RNA primers and replaces them with DNA.

• DNA ligase seals the gaps between Okazaki fragments.

Protein	Function
DNA polymerase	Synthesizes DNA
Primase	Synthesizes RNA primer
DNA ligase	Joins Okazaki fragments
Helicase	Unwinds DNA helix
SSBPs	Stabilize single strands
Topoisomerase	Relieves tension
Additional info: Table based on bacterial DNA replication proteins.

Replicating the Ends of Linear Chromosomes

The End Replication Problem and Telomeres

• Lagging strand synthesis leaves a single-stranded overhang at chromosome ends (telomeres).

• Without a solution, chromosomes would shorten with each division.

• Telomeres are repetitive, non-coding sequences at chromosome ends.

Role of Telomerase

• Telomerase extends the 3′ end using an RNA template it carries, allowing normal DNA synthesis to complete the lagging strand.

• Telomerase is active in gametes and stem cells, but not in most somatic cells.

• Telomere shortening limits cell division; cancer cells often reactivate telomerase for unlimited division.

Repairing Mistakes and DNA Damage

Accuracy of DNA Replication

• DNA polymerase is highly accurate, with an error rate of about one mistake per billion nucleotides.

• Correct base pairs are energetically favorable and have distinct shapes.

Proofreading and Mismatch Repair

• DNA polymerase proofreads and removes mismatched nucleotides using its exonuclease activity.

• Mismatch repair enzymes correct errors missed during replication by removing and replacing incorrect bases.

Repairing Damaged DNA

• DNA can be damaged by UV light, X-rays, and chemicals (e.g., thymine dimers from UV light).

• Nucleotide excision repair removes damaged DNA and fills in the correct sequence using the undamaged strand as a template.

DNA Repair Defects and Disease

• Xeroderma pigmentosum (XP): A rare autosomal recessive disease caused by mutations in nucleotide excision repair genes, leading to extreme UV sensitivity and increased cancer risk.

• Defects in DNA repair genes are often associated with cancer due to increased mutation rates.