series of experiments to prove that DNA was the genetic material
Alfred Hershey and Martha Chase in 1952
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)
virus and bacteria were…
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
Hershey and Chase demonstrated that
he 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
Rosalind Franklin and Maurice Wilkins used a method of X-ray diffraction to investigate the structure of DNA
- 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)
Franklin’s x-ray diffraction experiments demonstrated that…
DNA helix is both tightly packed and regular in structure
- Phosphates (and sugars) form an outer backbone and nitrogenous bases are packaged within the interior
Chargaff had also demonstrated that DNA is composed of…
an equal number of purines (A + G) and pyrimidines (C + T)
- This indicates that these nitrogenous bases are paired (purine + pyrimidine) within the double helix
- In order for this pairing between purines and pyrimidines to occur, the two strands must run in antiparallel directions
When Watson & Crick were developing their DNA model, they discovered that…
an A–T bond was the same length as a G–C bond
- Adenine and thymine paired via two hydrogen bonds, whereas guanine and cytosine paired via three hydrogen bonds
- If the bases were always paired this way, then this would describe the regular structure of the DNA helix (shown by Franklin)
Consequently, DNA structure suggests two mechanisms for DNA replication:
- Replication occurs via complementary base pairing (adenine pairs with thymine, guanine pairs with cytosine)
- Replication is bi-directional (proceeds in opposite directions on the two strands) due to the antiparallel nature of the strands
Helicase
- 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
DNA Gyrase
- 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
Single Stranded Binding (SSB) Proteins
- 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
DNA Primase
- 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
DNA Polymerase III
- 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)
DNA Polymerase I
- 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
DNA Ligase
- 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
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 polyermase occurs…
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
Leading versus Lagging Strands
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
