DNA Structure and Replication: A Comprehensive Guide

Posted on Aug 24, 2024 in Biology

Chapter 14: DNA Structure and Replication

Deoxyribonucleic Acid (DNA)

Deoxyribonucleic acid (DNA) is the molecule that serves as the genetic material for all living organisms. In 1868, Johann Miescher discovered “nuclein,” an acidic substance with high phosphorus content, within the nuclei of white blood cells. It took over 80 years to confirm that this material, now known as DNA, was the molecule responsible for inheritance. The DNA molecule provided an explanation for how genetic information is stored and replicated.

DNA as the Genetic Material

Several experiments demonstrated that DNA, not protein, carries genetic information.

Griffith’s Experiments

In 1928, Frederick Griffith identified a substance capable of genetically transforming bacteria, a process known as transformation.

Question: What is the nature of the genetic material?

Griffith studied two strains of Streptococcus pneumoniae:

  • Smooth strain (S): Highly infective (virulent), rapidly causing pneumonia and death in mice. The S form possesses a capsule that surrounds the cell, giving its colonies a smooth, shiny appearance on a laboratory dish.
  • Rough strain (R): Nonvirulent and does not kill mice. The R form lacks a capsule, resulting in colonies with a rough, nonshiny appearance.

Griffith injected the bacteria into mice and observed how the infections developed.

  1. Mice injected with live S cells (control): Mice die. Live S cells are found in their blood, confirming the virulence of S cells.
  2. Mice injected with live R cells (control): Mice live. No live R cells are found in their blood, confirming the nonvirulence of R cells. The capsule appears to be responsible for the virulence of the S strain.
  3. Mice injected with heat-killed S cells (control): Mice live. No live S cells are found in their blood, indicating that live S cells are necessary for virulence in mice.
  4. Mice injected with heat-killed S cells plus live R cells: Mice die. Live S cells are found in their blood, demonstrating that living R cells can be transformed into virulent S cells by a factor present in dead S cells.

Conclusion: Griffith concluded that molecules released upon the death of S cells could genetically alter living, nonvirulent R cells into the virulent S form. He termed this molecule the transforming principle and the process of genetic change transformation.

Avery’s Experiments

  • In the 1940s, Oswald Avery determined the chemical nature of Griffith’s transforming principle.
  • Avery broke down heat-killed S bacteria and destroyed one class of molecules at a time: protein, DNA, or RNA.
  • Destroying proteins or RNA did not prevent the extract from transforming R bacteria into virulent S bacteria.
  • Destroying DNA prevented transformation, indicating that DNA was the transforming principle.

Hershey and Chase’s Experiments

Hershey and Chase studied bacteriophages (or phages), viruses that infect the bacterium E. coli.

Virus
  • An infectious agent composed of DNA or RNA enclosed in a protein coat.
  • Reproduces only within a host cell, utilizing host materials.

In 1952, Alfred Hershey and Martha Chase definitively proved that DNA is the hereditary molecule.

A phage infects a bacterium by attaching to its surface. The T2 phage used by Hershey and Chase consists solely of a DNA core surrounded by proteins. They radioactively labeled either the DNA with 32P or the protein with 35S and tracked the molecule. Their results showed that labeled DNA, not labeled protein, entered the cell and appeared in progeny phages.

Conclusion:

  • The experiments of Griffith, Avery and his colleagues, and Hershey and Chase established DNA, not proteins, as the carrier of genetic information.
  • This research also established the term transformation: the alteration of a cell’s hereditary type through the uptake of DNA released from another cell’s breakdown.

DNA Structure

DNA comprises four different nucleotides, each consisting of:

  1. One five-carbon sugar (deoxyribose)
  2. One phosphate group
  3. One of four nitrogenous bases (A, G, T, C)
  • Adenine (A) and guanine (G) are purines, built from a pair of fused carbon-nitrogen rings.
  • Thymine (T) and cytosine (C) are pyrimidines, built from a single carbon-nitrogen ring.

Chargaff’s Rules

Erwin Chargaff discovered that nitrogenous bases in DNA exist in specific ratios.

  • The amount of purines equals the amount of pyrimidines.
  • The amount of adenine equals the amount of thymine.
  • The amount of guanine equals the amount of cytosine.

Chargaff’s rule: A = T, and G = C

DNA Nucleotides and Polynucleotide Chains

Deoxyribose sugars are connected by phosphate groups in an alternating pattern, creating a sugar-phosphate backbone. Each phosphate group links the 3′ carbon of one sugar to the 5′ carbon of the next sugar. This linkage is called a phosphodiester bond.

The DNA polynucleotide chain has polarity. One end has a phosphate group bound to the 5′ carbon of a deoxyribose sugar (the 5′ end), while the other end has a hydroxyl group bound to the 3′ carbon of a deoxyribose sugar (the 3′ end).

X-Ray Diffraction and the DNA Model

Maurice Wilkins and Rosalind Franklin, at King’s College, London, independently used X-ray diffraction to study DNA structure.

James Watson and Francis Crick, using data from Franklin’s X-ray diffraction images, developed their groundbreaking 1953 model for DNA structure, revolutionizing the biological sciences.

In Watson and Crick’s model, the purine-pyrimidine base pairs (A-T and G-C) are held together by hydrogen bonds—two between A and T and three between G and C. Due to hydrogen bonding requirements, C cannot pair with A, and G cannot pair with T.

The two strands of a DNA molecule are complementary to each other, following complementary base pairing rules:

  • An A on one strand always pairs with a T on the opposite strand.
  • A G on one strand always pairs with a C on the opposite strand.

Watson, Crick, and Wilkins shared the Nobel Prize for their discovery in 1962. Rosalind Franklin, unfortunately, died of cancer in 1958 at the age of 38.

DNA Replication

DNA replication is semi-conservative. Each new double helix consists of one old strand, originating from the parental DNA molecule, and one newly synthesized strand.

The process of DNA replication involves the following steps:

  1. The two strands of the DNA molecule unwind.
  2. DNA polymerase adds nucleotides to an existing chain.
  3. New synthesis proceeds in the 5′→3′ direction, antiparallel to the template strand.
  4. Nucleotides are added to the newly synthesized chain according to the A-T and G-C complementary base-pairing rules.

Due to the antiparallel nature of the DNA molecule, only one template strand runs in a direction that allows DNA polymerase to create a continuous 5′→3′ copy. This strand is called the leading strand.

DNA polymerase copies the other strand in short segments called Okazaki fragments. These fragments are synthesized in the direction opposite to DNA unwinding (discontinuous replication). This strand is called the lagging strand.

Proofreading Mechanism

DNA polymerases possess a proofreading mechanism that allows them to backtrack and remove incorrectly paired nucleotides.

Enzymes in DNA Replication

Numerous enzymes work together to replicate DNA:

  • DNA helicase unwinds DNA.
  • Primase initiates the synthesis of all new strands.
  • DNA polymerase III is the primary polymerase.
  • DNA polymerase I synthesizes the lagging strand.
  • DNA ligase joins Okazaki fragments together.