Griffith, Avery, Hershey-Chase Experiments

• Frederick Griffith in 1928 questioned whether proteins or DNA carried genetic information.
• R strain Streptococcus pneumoniae is non-deadly and non-pathogenic.
• S strain Streptococcus pneumoniae is deadly and pathogenic.
• Heat-killed S strain cells by themselves do not cause death.
• A mix of heat-killed S strain and live R strain results in death, indicating a "transforming principle".

Oswald Avery's Discoveries

• Oswald Avery and colleagues in 1944 investigated the transforming principle.
• They treated heat-killed S cells with protease, RNAse, and DNAse.
• Samples treated with protease and RNAse transformed R cells, killing the mouse.
• Samples treated with DNAse did not transform R cells, and the mouse lived.
• Experiments showed that DNA is the holder of genetic information, but further confirmation was requested.

Alfred Hershey and Martha Chase Experiment

• Alfred Hershey and Martha Chase's 1952 blender experiment further confirmed DNA as genetic material.
• Proteins contain CHONS, while DNA contains CHONP.
• Bacteriophages were grown in radioactive Sulfur (S) and Phosphorus (P).
• Bacteria infected with bacteriophages produce more bacteriophages.
• Blending and centrifugation separated cells from phage coats.
• Radioactive P was found inside bacteria and in new bacteriophages.
• Radioactive S remained outside the cells.

Erwin Chargaff's Contribution

• Erwin Chargaff stated in 1950 that the number of purine nucleotides equals the number of pyrimidine nucleotides.
• Adenine pairs with Thymine (A-T), and Cytosine pairs with Guanine (C-G).

Discoveries of Maurice Wilkins, Rosalind Franklin, James Watson, and Francis Crick

• Rosalind Franklin used X-ray crystallography and found the dimensions of DNA strands and the helical structure.
• James Watson and Francis Crick created the first accurate DNA structure model in 1953.
• Nucleotides consist of phosphate, a 5-carbon sugar, and a nitrogen base.
• DNA has a double helix structure.
• The sugar-phosphate backbone is formed by phosphodiester bonds.
• Complementary base pairs (A-T and C-G) are held together by hydrogen bonds.
• DNA strands are antiparallel, running in opposite directions (3' → 5' and 5' → 3').

DNA Replication

• Bacteria was grown in N15 medium causing it to incorporate into its DNA, then the bacteria was moved to N14 medium.
• DNA made after the switch would have to be made up of N14.
• Centrifugation was used to sort DNA by density after each generation.

Models of Replication

• Conservative replication results in both original DNA strands together and two new strands together.
• Semi-conservative replication results in two DNA molecules, each with one original strand and one new strand.
• Dispersive replication results in each strand containing a patchwork of original and new DNA.

Results

• After generation 1, DNA of intermediate weight ruled out the conservative model.
• After generation 2, DNA that was both light and intermediate in weight evidenced the semi-conservative model.

Details of DNA Replication

• Replication occurs rapidly and simultaneously at multiple locations via replication bubbles.
• Nucleotides are added to the 3' end; they cannot be added to the 5' end.
• The leading strand is built continuously, while the lagging strand is built in fragments.
• Okazaki fragments are short DNA sections formed during discontinuous synthesis of the lagging strand.

Initiation

• Gyrase/Topoisomerase cleaves the DNA helix while relieving tension.
• Helicase breaks the hydrogen bonds between complementary bases.
• The unzipped helix ends at the replication fork.
• Replication origins are A-T rich regions (easier to break due to only 2 bonds).
• Single-stranded binding proteins (SSB proteins) prevent separated template strands from reattaching.
• Primase lays RNA primers at the 5' end.
• DNA Polymerase III adds the correct deoxyribonucleotide triphosphate (dNTP) to the 3' end, using the energy in phosphate bonds.
• Synthesis occurs in the 5' to 3' direction

Leading vs Lagging Strand

• The leading strand synthesizes continuously, involving one DNA polymerase.
• The lagging strand synthesizes discontinuously, forming Okazaki fragments, and involves multiple polymerases.
• DNA Polymerase I removes RNA primers and replaces them with the correct DNA.
• DNA Ligase joins Okazaki fragments through phosphodiester bonds.
• DNA Polymerase and exonucleases proofread and replace incorrect nucleotides.