LECTURE 13 DNA

Describe evidence that DNA carries genetic information

Frederick Griffith (1928) performed studies using two strains of the Streptococcus pneumoniae bacterium - one pathogenic, the other non-pathogenic. Pathogenic bacteria injected into a mouse caused the mouse to die, while non-pathogenic bacteria and heat-killed pathogenic bacteria did not affect the mouse. However, when Griffith mixed together heat-killed pathogenic cells and non-pathogenic cells, the mouse died, and living pathogenic cells could be found in the mouse's blood. This suggested that the living non-pathogenic cells were inheriting nformation from the dead pathogenic cells. Later work by other scientists identified the transforming substance as DNA.

Hershey and Chase's studies (1952) showed further evidence that DNA carries genetic information with their studies of viruses and E. coli. Hershey and Chase incorporated radioactive sulfur atoms into the protein of the bacteriophage and radioactive phosphorus into its DNA to see if it is proteins or DNA that enters the E. coli. They found that DNA entered the cell while proteins did not, and after some time the E. coli released new phages that contained radioactive phosphorus. Hershey and Chase concluded that it was DNA that was carrying genetic information to allow the cells to produce new viral DNA and proteins.

In 1950, Erwin Chargaff discovered that the base composition of DNA varies between different species, further suggesting that DNA is the genetic material.

Show that DNA is an anti-parallel double-stranded polynucleotide in which base-pairing plays a crucial role

DNA consists of two anti-parallel (run in opposite directions, 5'-3' and 3'-4') strands made up of a sugar-phosphate backbone and nitrogenous bases joining the two strands, twisted into a double helix. One sugar-phosphate molecule joined to a nitrogenous base makes up one nucleotide. The sugar-phosphate molecules are joined to each other by covalent bonds (phosphodiester bonds), and the base pairs are joined to each other by hydrogen bonds. Bases A and G are purines (double-ringed), and bases C and T are pyrimidines (single-ringed). A bonds with T (2 H-bonds) and G bonds with C (3 H-bonds). Evidence can be seen in that there are equal ratios of A and T bases and G and C bases in DNA.

https://www.youtube.com/watch?v=xozXUIVeSpY&list=PLU9wEyzVsaZXPYJKqKM7qyb4D-vosDwAn

Explain the Meselson-Stahl experiment to demonstrate semi-conservative replication of DNA

Meselson and Stahl cultured E. coli over several generations in a culture containing nitrogen isotope 15N. They then transferred the bacteria to a culture containing only 14N, which is lighter than 15N. They extracted DNA from samples taken after the first and second DNA replication and centrifuged the DNA samples to separate the DNA by density. The sample taken after the first replication showed one band of hybrid 15N and 14N DNA, while the sample taken after the second replication showed both light DNA and hybrid DNA. This concluded that DNA replication is semi-conservative.

Discover that DNA polymerase and other proteins achieve DNA replication

DNA replication: https://www.youtube.com/watch?v=NX9kwdQ3R6s; https://www.youtube.com/watch?v=UizoPujYRuU

Topoisomerase: breaks, swivels, and rejoins parental DNA ahead of replication fork, relieving strain caused by unwinding.

Helicase: unwinds DNA strands.

Single-strand binding protein: binds to and stabilises a single-stranded DNA until it is used as a template.

Primase: synthesises RNA primers on the lagging strand.

DNA polymerase III: synthesises new DNA strand by adding nucleotides in a 5' to 3' direction.

DNA polymerase I: replaces RNA primers with DNA on lagging strand.

DNA ligase: forms bond between DNA fragments

Explain that DNA replication is a very accurate process but that rare mistakes can lead to mutations

DNA polymerase proofreads nucleotides added to the template strand during the process of building the new strand. If the polymerase detects a mismatched nucleotide, it removes the nucleotide before continuing synthesis. In the case that the mismatched nucleotide evades proofreading by polymerase, it can be removed by other enzymes that replace incorrectly matched nucleotides. DNA nucleases cut out (excise) mismatched base pairs and the resulting gap is filled in with nucleotides by DNA polymerase and ligase. DNA molecules can be altered after replication by harmful chemical and physical agents (eg X-rays) and can change under normal cellular conditions, and thus constant care is required.

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Lectures 13-15