7.1 DNA STRUCTURE
In 1952, Alfred Hershey and Martha Chase conducted a series of experiments to
prove that DNA was the genetic material. 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.
P–viruses
The bacterial pellet was found to be radioactive when infected by the 32
(DNA) but not the S–viruses (protein). This demonstrated that DNA, not protein,
35
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)
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.
DNA replication is a semi-conservative process that is carried out by a complex
system of enzymes
, 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.
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.
In 1952, Alfred Hershey and Martha Chase conducted a series of experiments to
prove that DNA was the genetic material. 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.
P–viruses
The bacterial pellet was found to be radioactive when infected by the 32
(DNA) but not the S–viruses (protein). This demonstrated that DNA, not protein,
35
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)
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.
DNA replication is a semi-conservative process that is carried out by a complex
system of enzymes
, 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.
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.
