Search for Genetic Material

The Search for Genetic Material

• Early 1900's main question: What is genetic information made of, proteins or nucleotides?
• Key experiments led to the current understanding of genetic material structure
• In 1928, Frederick Griffith studied pneumonia bacterium in mice
• Griffith studied virulent (S strain) and non-virulent (R strain) pneumonia bacteria

Griffith's Experiment

• Griffith's experiment in 1928: Bacterial transformation, which is the transfer of DNA to another strain
• Griffith's experiment provided the first hint that proteins were not the genetic material
• Griffith concluded living R bacteria transformed into pathogenic S bacteria via an unknown, heritable substance from dead S cells

Further Key Experiments

• Avery's experiment in 1942 showed transformation only happens if DNA is intact
• Schrodinger's experiment in 1944 stated genetics are key to life
• Schrodinger determined a gene is made of different individual subunits

DNA Composition

• In the time period there was knowledge that DNA is made of pyrimidines (C and T) and purines (A and G)
• Purines were known to be wider than pyrimidines

Chargaff's Rule

• In 1947, Chargaff determined that base composition varies between species
• In a species, the number of A:T bases are equal, and the number of G:C bases are equal

Hershey-Chase Experiment

• The 1952 Hershey and Chase experiment used T2 bacteriophage, a virus infecting E. coli
• The main question: Is protein or DNA responsible for reprogramming bacteria to make new viruses?

Rosalind Franklin and X-Ray Crystallography

• In 1953, Rosalind Franklin and Maurice Wilkins used X-ray crystallography to study molecular structure
• Rosalind Franklin produced an X-ray diffraction image of DNA, crucial for deducing its structure
• The spots in Franklin's photograph indicated diffraction from X-rays passing through purified DNA fibers

Watson and Crick's Discovery

• In 1953, James Watson, an American, visited Francis Crick in Cambridge
• Watson saw Franklin's X-ray diffraction image and knew what X-ray diffraction pattern helical models produce
• Watson & Crick determined DNA was a double helix

Watson and Crick Model

• In 1953, Watson and Crick created the double-helix model
• They used Chargaff's base pairing findings
• Genetic information is carried on the base sequence in the double helix model

DNA Basics

• DNA is made of nucleotides
• Nucleotides include a nitrogenous base that is hydrophobic
• Nucleotides include a 5C pentose sugar, specifically deoxyribose
• Nucleotides contain a phosphate group

DNA Structure

• Complementary base pairs in DNA are A + T and G + C
• Each strand is a complementary copy of the other
• DNA is held together by hydrogen bonds
• DNA codes for protein synthesis as well as acting as a template for RNA synthesis

DNA Charge

• DNA is negatively charged

DNA Replication

• Watson and Crick were interested in how DNA replicated itself
• The structure suggested the basic mechanism of replication
• Watson and Crick hypothesized replication is semi-conservative: parent strands separate to provide two templates for the synthesis of 2 new strands

Semi-Conservative Replication

• Each new strand is composed of one 'old' and one 'new' strand

Alternative Models of DNA Replication

• DNA replication could also be conservative or dispersive
• Experiments were required to confirm which model DNA replication follows

Meselson and Stahl Experiment

• A clever experiment that tested if DNA replicated using conservative, semi-conservative, or dispersive method

DNA Replication Process

• Replication origin: the site where DNA replication begins
• Eukaryotes may have hundreds of replication origins, while bacteria usually have only one

Elongating New DNA

• Elongation of new DNA happens at a replication fork at the 3' end of a growing strand
• The new strand always grows from 5' to 3'

Replication Enzymes and Process

• Helicase unwinds the double helix and starts replication
• Single-strand binding protein stabilizes single-stranded DNA
• Primase creates an RNA primer/attaches to an initiation point of a DNA strand
• DNA Polymerase catalyzes the synthesis of a new DNA strand
• DNA ligase joins Okazaki fragments of 3'-5' strand

Role of DNA Polymerase

• DNA polymerase produces a complimentary strand from 5' to 3' direction
• Leading and lagging strands occur from slightly different methods of synthesis

Proofreading and Repairing DNA

• DNA mismatch may be caused by errors in replication or UV light, which causes thymine dimers
• Repairs are done by DNA polymerase II, which is much slower than DNA pol III
• The bad bit of DNA is cut out by a DNA-cutting enzyme called nuclease
• Ligase seals the new piece of DNA together

Types of DNA Polymerases

• DNA Polymerase I replaces RNA primers
• DNA Polymerase II repairs mutated nucleotides
• DNA Polymerase III is the main DNA polymerase
• These DNA polymerases are bacterial, and eukaryotic organisms have ~16 DNA polymerases with various functions

Replicating DNA Molecule Ends

• The ends of eukaryotic chromosomal DNA get shorter with each division
• The end of the DNA lagging strand is not replicated

Telomeres

• Telomeres are end sequences on DNA that are not essential for cell function
• Telomeres are lost slowly as cells divide

Role of Telomerase

• Telomerase replaces the lost bit at the end of the 5' end of telomeres
• Telomerase also contains a section of RNA

Telomerase and Health

• Shortening may protect cells from cancer by limiting number of divisions, but also results in aging
• Cells from large tumors have unusually short telomeres due to many divisions
• Telomerase activity found in cancer cells, telomerase can stabilize telomere length and allow cancer to persist

DNA Packaging

• Chromatin is a complex of DNA and protein
• Chromatin changes during the cell cycle
• Heterochromatin, areas existing in highly condensed state
• Euchromatin is less compact and more dispersed