DNA Structure and Genetic Material

• Nucleic acids determine structure and replication.
• The genetic material was identified biochemically, focusing on nucleic acid structure and DNA replication.

Genetic Material Criteria

• Genetic material stores necessary information to construct an entire organism.
• It passes from parent to offspring and divides during cell division (transmission).
• Genetic material copies accurately during replication.
• It accounts for the known variation within and between species.

DNA or Protein?

• The Griffith experiment involved bacterial transformation.
• Streptococcus pneumoniae bacteria was injected into mice.
• Two strains of bacteria were injected, S strain and R strain.
  • S strain produces shiny and smooth colonies and is encapsulated.
  • R strain produces rough appearance colonies and is non-encapsulated.
• When injected into mice, S strain killed the mice (virulent) due to the capsule.
• When injected into mice, R strain did not kill the mice (non-virulent).
• Heat-killed virulent S strain injected into mice did not kill the mice.
• A combination of heat-killed virulent S strain and live non-virulent R strain killed the mice.
• Living S strain was recovered from the dead mouse.
• A substance necessary for virulence passed from dead S strain to living R strain bacteria, meaning the R strain bacteria was transformed.
• The transforming substance was identified as genetic material.
• Using T2 virus that infects Escherichia coli identified DNA as genetic material
  • The virus is a Bacteriophage (phage).
  • The Phage coat made entirely of protein has DNA inside the capsid

Hershey-Chase Experiment

• In 1952, researchers studied the T2 virus infecting Escherichia coli.
• Chemical differences between DNA and protein include:
  • DNA has phosphate.
  • Protein has sulfate.
• Hershey and Chase used radioactive 32P to label the DNA of the phage and radioactive 35S to label the protein of the phage.
• Viral DNA (not protein) enters the host, therefore DNA is the genetic material.

DNA Structure

• Deoxyribonucleic Acid (DNA) contains 4 types of nucleotides, each possessing a nitrogen-containing base, a phosphate group (phosphoric acid), and a pentose sugar.
• Two strands form a double helix.
• The conventional numbering system is conventional in DNA.
• Sugar carbons are numbered 1' to 5'.
• The base attaches to the 1' carbon.
• The phosphate attaches to the 5' carbon.
• DNA contains two strands with purine bases (double ring): Adenine (A) and Guanine (G).
• It also contains two strands with pyrimidine bases (single ring): Thymine (T) and Cytosine (C).
• Nucleotides covalently bond.
• A phosphodiester bond is the phosphate group links 2 sugars.
• Phosphates and sugars form the backbone.
• Bases project from the backbone.
• The directionality is 5' to 3'.
• An example sequence: 5' – TACG – 3'

DNA Structure Solutions

• The building blocks are nucleotides in both DNA and RNA are nucleotides.
• A DNA (or RNA) strand is a string of nucleotides.
• Two strands twist to form a double helix.
• DNA in cells associate with different proteins to form chromosomes.
• A genome is the complete complement of genetic material of an organism.
• James Watson and Francis Crick proposed the structure of the DNA double helix in 1953 with Maurice Wilkins.
  • They used Linus Pauling's method of working out protein structures using simple ball and stick models.
• Rosalind Franklin's X-ray diffraction results provided crucial information.
• Erwin Chargaff analyzed base composition of DNA, which also provided important information.
• Watson and Crick put together the pieces of information and with data consistent with the ball-and-stick model, and were awarded the Nobel Prize in 1962.
• Rosalind Franklin died and the Nobel is not awarded posthumously.
• DNA is normally double stranded and helical
• DNA contains a sugar-phosphate backbone with bases on the inside.
• DNA stablized by H-bonding.
• DNA has base pairs with specific pairing.
• AT/GC pairing consistent with Chargoff's rule includes:
  • A pairs with T.
  • G pairs with C.
• There are 10 base pairs per turn.
• 2 DNA strands are complementary.
  • For example, 5' – GCGGATTT – 3' would match to 3' – CGCCТААА – 5'.
• 2 strands are antiparallel: One strand is 5' to 3', while the other is 3' to 5'.

Chargaff's Rules

• Edwin Chargaff demonstrated in 1951, that the 4 nucleotides (A, C, G & T) are not equally present in the DNA and that the ratio varies a lot between species.
• In each species, the amount of A=T and the amount of G=C.
• The percentage of A+G equals 50% and the percentage of T+C equals 50%

DNA Replication

• Three schemes are described for DNA replication including:
  • Dispersive
  • Semiconservative
  • Conservative
• The Meselson-Stahl experiment of 1958 helped to elucidate replication.
• Each old strand of DNA serves as a template for a new strand (Semiconservative replication).
• One old strand is conserved in each daughter molecule.
• During replication, 2 parental strands separate and serve as template strands.
• New nucleotides must obey the AT/GC rule (Chargaff's Rules).
• End result 2 new double helices with same base sequence as the original

Replication Points

• Replication begins at the point referred to a the origin of replication which is the starting point for replication.
• Bidirectional replication allows replication to proceeds outward in opposite directions
• Bacteria have a single origin for bidirectional replication.
• Eukaryotes require multiple origins for bidirectional replication
• Origin of replication provides an opening, called a replication bubble that forms two replication forks.
• DNA replication occurs near the fork.
• Synthesis begins with a primer and proceeds 5' to 3'.
• The leading strand is made in the direction the fork is moving and synthesized as one continuous molecule.
• The lagging strand contains Okazaki fragments that must be connected later.
• Helicase binds to DNA and travels 5' to 3' using Adenosine Triphosphate to separate the strand and move the fork forward and unwinds or "unzips" hydrogen bonds which is unzipping
• Topoisomerase relieves additional coiling ahead of replication fork and DNA.
• Parental strands are kept open to act as templates by the Single-strand binding proteins.
• Polymerase covalently links nucleotides using complementary base paring to form new strands.
• New "daughter" DNA has 1 old and 1 new strand

Key Features

• Free nucleotides consist of 3 phosphate groups which when broken releases a covalent bond of pyrophosphate providing energy to connect adjacent nucleotides.
• DNA polymerase's 2 enzymatic features are important for the leading and lagging strands.
  • DNA polymerase is unable to begin DNA synthesis on a bare template strand.
  • DNA primase must make a short RNA primer that will later be removed and replaced with DNA.
  • DNA polymerase can only work 5' to 3' (directional synthesis).

DNA Replication Process

• In the leading strand, DNA primase makes one RNA primer, and DNA polymerase attaches nucleotides in a 5' to 3' direction as it slides forward.
• In the lagging strand:
  • DNA is synthesized 5' to 3' but in a direction away from the fork.
  • Okazaki fragments create a short RNA primer made by DNA primase at the 5' end.
  • The. DNA is then laid down by DNA polymerase.
  • The RNA primers will be removed by DNA polymerase and filled in with DNA.
  • DNA ligase will join adjacent DNA fragments.
• Three reasons for replication accuracy include:
  • H-bonding between A and T, or G and C is more stable than mismatches.
  • The active site of DNA polymerase is unlikely to form bonds if pairs are mismatched.
  • DNA polymerase removes mismatched pairs.
    • "Proofreading" results in DNA polymerase backing up and digesting linkages.
    • Other DNA repair enzymes.

Explore the double helix structure of DNA, its components like sugars and bases, and key genetic concepts. Learn about DNA replication accuracy. Understand experiments that revealed DNA's role.