UNIT 7.1 Flashcards
Explain the process of the Hershey & Chase (1952) Experiment
Viruses (T2 bacteriophage) were grown in one of two isotopic mediums in order to radioactively label a specific viral component
Viruses grown in radioactive sulfur (35S) had radiolabelled proteins (sulfur is present in proteins but not DNA)
Viruses grown in radioactive phosphorus (32P) had radiolabeled DNA (phosphorus is present in DNA but not proteins)
The viruses were then allowed to infect a bacterium (E. coli) and then the virus and bacteria were separated via centrifugation
The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant
The bacterial pellet was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein)
This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria
Explain the process of the structure of DNA (Wilkins & Franklin) Experiment
DNA was purified and then fibres were stretched in a thin glass tube (to make most of the strands parallel)
The DNA was targeted by a X-ray beam, which was diffracted when it contacted an atom
The scattering pattern of the X-ray was recorded on a film and used to elucidate details of molecular structure
From the scattering pattern produced by a DNA molecule, certain inferences could be made about its structure
- Composition: DNA is a double stranded molecule
- Orientation: Nitrogenous bases are closely packed together on the inside and phosphates form an outer backbone
- Shape: The DNA molecule twists at regular intervals (every 34 Angstrom) to form a helix (two strands = double helix)
How many hydrogen bonds are in a;
A-T bond ?
G-C bond?
A-T = 2
G-C = 3
However, Both bonds are the same length.
DNA can be described as what kind of process ?
Semi - Conservative (Demonstrated by Meselson-Stahl)
What is the role of Helicase in DNA Replication?
Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs.
This occurs at specific regions (origins of replication), creating a replication fork of two strands running in antiparallel directions.
What is the role of DNA Gyrase in DNA Replication?
DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase.
It does this by relaxing positive supercoils (via negative supercoiling) that would otherwise form during the unwinding of DNA.
What is the role of Single stranded binding proteins in DNA Replication?
SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing.
These proteins also help to prevent the single stranded DNA from being digested by nucleases.
SSB proteins will be dislodged from the strand when a new complementary strand is synthesised by DNA polymerase III.
What is the role of DNA Primase in DNA Replication?
DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template strands.
The RNA primer provides an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one.
What is the role of DNA Polymerase III in DNA Replication?
Free nucleotides align opposite their complementary base partners (A = T ; G = C)
DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
On the leading strand, DNA pol III is moving towards the replication fork and can synthesise continuously
On the lagging strand, DNA pol III is moving away from the replication fork and synthesises in pieces (Okazaki fragments)
What is the role of DNA Polymerase I in DNA Replication?
As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides
What is the role of DNA Ligase in DNA Replication?
DNA ligase joins the Okazaki fragments together to form a continuous strand
It does this by covalently joining the sugar-phosphate backbones together with a phosphodiester bond
Explain the purpose of Okazaki Fragments in DNA Replication
- DNA polymerase cannot initiate replication, it can only add new nucleotides to an existing strand
- For DNA replication to occur, an RNA primer must first be synthesised to provide an attachment point for DNA polymerase
- DNA polymerase adds nucleotides to the 3’ end of a primer, extending the new chain in a 5’ → 3’ direction
-Free nucleotides exist as deoxynucleoside triphosphates (dNTPs) – they have 3 phosphate groups
DNA polymerase cleaves the two additional phosphates and uses the energy released to form a phosphodiester bond with the 3’ end of a nucleotide chain
Outline the differences between the leading and lagging strand
Because double-stranded DNA is antiparallel, DNA polymerase must move in opposite directions on the two strands
On the leading strand, DNA polymerase is moving towards the replication fork and so can copy continuously
On the lagging strand, DNA polymerase is moving away from the replication fork, meaning copying is discontinuous
As DNA polymerase is moving away from helicase, it must constantly return to copy newly separated stretches of DNA
This means the lagging strand is copied as a series of short fragments (Okazaki fragments), each preceded by a primer
The primers are replaced with DNA bases and the fragments joined together by a combination of DNA pol I and DNA ligase
What is DNA sequencing ?
DNA sequencing refers to the process by which the base order of a nucleotide sequence is elucidated
What are Dideoxynucleotides ?
Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group necessary for forming a phosphodiester bond
Consequently, ddNTPs prevent further elongation of a nucleotide chain and effectively terminate replication
