DNA and the Gene: Synthesis and Repair – Study Notes

DNA and the Gene: Synthesis and Repair

Learning Outcomes

• Describe the structure of DNA

• Understand experimental evidence for DNA as genetic material and the semiconservative nature of replication

• Explain the process of DNA replication and the roles of major enzymes

• Distinguish between leading and lagging strand synthesis

• Describe the end-replication problem and its solution

• Explain proofreading and DNA repair mechanisms

The Search for the Genetic Material

DNA vs. Protein as Genetic Material

• Early 20th-century research established that genes are located on chromosomes, which are composed of DNA and protein.

• Due to the apparent simplicity of DNA (only four nucleotide types), protein was initially favored as the genetic material.

• Key experiments (Hershey-Chase, Chargaff) provided strong evidence for DNA as the genetic material.

The Hershey-Chase Experiment

• Used bacteriophage T2 to infect Escherichia coli (E. coli).

• Phages were labeled with radioactive isotopes: 35S for protein, 32P for DNA.

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

• Conclusion: DNA, not protein, carries genetic information.

Structure of a Phage (Bacterial Virus)

• Bacteriophages (phages) are viruses that infect bacteria.

• Phage structure: protein capsid (shell) and DNA core.

• After infection, the capsid remains outside, and only DNA enters the host cell.

Primary Structure of DNA

Nucleotide Composition

• DNA is a polymer of nucleotides, each consisting of:

  • A phosphate group

  • A deoxyribose sugar

  • A nitrogenous base (Adenine [A], Thymine [T], Cytosine [C], Guanine [G])

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

Chargaff's Rules

• DNA composition varies between species.

• In any species, the amount of A = T and G = C.

• These findings were crucial for understanding DNA's double helix structure.

Deciphering DNA Structure

Key Scientists

• James Watson and Francis Crick: Proposed the double helix model.

• Rosalind Franklin: Provided critical X-ray crystallography data.

• Maurice Wilkins: Collaborated with Franklin and shared data with Watson and Crick.

• Linus Pauling: Incorrectly proposed a triple helix model.

Base Pairing in DNA

• Watson and Crick determined that:

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

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

• Other pairings are structurally incompatible.

• This base pairing explains Chargaff's rules.

Double Helix and Antiparallel Strands

• DNA consists of two antiparallel strands (one runs 5'→3', the other 3'→5').

• The double helix is stabilized by hydrogen bonds and base stacking interactions.

• Distance between bases: 0.34 nm; one full turn: 3.4 nm (10 base pairs per turn).

DNA Replication

Semiconservative Replication

• Each new DNA molecule consists of one parental (old) strand and one newly synthesized strand.

• Alternative models (conservative, dispersive) were disproven by the Meselson-Stahl experiment.

The Meselson-Stahl Experiment (1958)

• Used E. coli grown in heavy (15N) and light (14N) nitrogen media.

• After replication, DNA showed intermediate density, consistent with semiconservative replication.

Origins of Replication

• Replication begins at specific sites called origins of replication.

• E. coli has a single origin; eukaryotes have multiple origins per chromosome.

• Replication proceeds bidirectionally, forming replication bubbles with two forks.

Mechanism of DNA Replication

Enzymes and Proteins Involved

• DNA polymerase: Synthesizes new DNA by adding nucleotides to a primer strand.

• Primase: Synthesizes short RNA primers to provide a starting point for DNA polymerase.

• Helicase: Unwinds the DNA double helix at the replication fork.

• Single-strand binding proteins (SSB): Stabilize unwound DNA strands.

• Topoisomerase: Relieves supercoiling ahead of the replication fork.

• Ligase: Joins Okazaki fragments on the lagging strand.

Leading and Lagging Strands

• DNA polymerase can only add nucleotides to the 3' end of a growing strand (5'→3' direction).

• Leading strand: Synthesized continuously toward the replication fork.

• Lagging strand: Synthesized discontinuously away from the fork in short segments called Okazaki fragments.

Summary Table: Major Enzymes in DNA Replication

Enzyme/Protein	Function
DNA Polymerase III	Main enzyme for leading and lagging strand synthesis
Primase	Synthesizes RNA primers
Helicase	Unwinds DNA helix
SSB Proteins	Stabilize single-stranded DNA
Topoisomerase	Relieves supercoiling
DNA Polymerase I	Removes RNA primers, replaces with DNA
Ligase	Joins Okazaki fragments

End Replication Problem and Telomeres

End Replication Problem

• Linear chromosomes cannot be fully replicated at the 3' ends by DNA polymerase, leading to progressive shortening.

Telomeres and Telomerase

• Telomeres: Repetitive, non-coding DNA sequences at chromosome ends (e.g., TTAGGG in humans).

• Telomerase: An enzyme that extends telomeres using an RNA template, preventing loss of genetic information in germ cells and some stem cells.

• Most somatic cells lack telomerase activity, leading to gradual telomere shortening and cellular aging.

• Cancer cells often reactivate telomerase, enabling unlimited division.

Proofreading and DNA Repair

Proofreading by DNA Polymerase

• DNA polymerases have 3'→5' exonuclease activity to remove incorrectly paired nucleotides.

• This proofreading reduces the error rate to about 1 in 107 nucleotides.

Mismatch and Excision Repair

• Mismatch Repair (MMR): Corrects errors missed by proofreading.

• Base Excision Repair (BER): Removes and replaces damaged bases.

• Nucleotide Excision Repair (NER): Removes bulky lesions (e.g., thymine dimers caused by UV light).

• Defects in repair systems can lead to diseases (e.g., Xeroderma pigmentosum, hereditary cancers).

Summary Table: DNA Repair Mechanisms

Repair Mechanism	Type of Damage	Key Features
Proofreading	Incorrect base insertion	3'→5' exonuclease activity of DNA polymerase
Mismatch Repair	Mismatched bases	Removes and replaces incorrect bases post-replication
Base Excision Repair	Small, non-helix-distorting base lesions	Removes damaged base, replaces with correct nucleotide
Nucleotide Excision Repair	Bulky, helix-distorting lesions (e.g., thymine dimers)	Removes short single-stranded DNA segment containing lesion

Key Equations and Concepts

• Phosphodiester bond formation:

  • $\text{(DNA strand)}_{n} + \text{dNTP} \rightarrow \text{(DNA strand)}_{n+1} + \text{PP}_i$

• Base pairing: $\text{A} = \text{T},\ \text{G} = \text{C}$

Example Application

• Replication of a template strand 3'-AAGTCAGT-5' yields a complementary strand 5'-TTCAGTCA-3'.

• Semiconservative replication means each daughter DNA has one old and one new strand.

Summary

• DNA is the hereditary material, structured as a double helix with specific base pairing.

• Replication is semiconservative, involving a complex set of enzymes and proteins.

• Proofreading and repair mechanisms ensure high fidelity of genetic information.

• Telomeres and telomerase solve the end-replication problem in eukaryotes.