Chapter 7: Nucleic Acids
7.1: DNA Structure and Replication
How does the huge DNA fit into a much smaller cell in our
organisms? More than 50% of a chromosome is built of protein and the bulk of
these proteins has a support and packaging role. The packaging of DNA is
important because it makes sure that those excessive DNA are able to fit nicely in
a cell that is many times smaller. This packaging is achieved by coiling the DNA
double helix and looping it around protein beads called nucleosomes.
In eukaryotic organisms, the DNA is packaged with histone proteins to create a
compacted structure called a nucleosome
● nucleosomes help to supercoil the DNA, resulting in a greatly compacted
structure that allows for more efficient storage
● Supercoiling helps to protect the DNA from damage and also allows
chromosomes to be mobile during mitosis and meiosis.
Hershey and Chase experiment:
In the mid-twentieth century, scientists were still unsure if the DNA (core) or
proteins (coat) contain genetic material. This is because 50% of chromosomes
consist of proteins. How
do we know now for sure that the DNA
contains the genetic material and not proteins? Hershey and Chase
conducted an experiment which aimed to prove that DNA contains the genetic
code.
Hershey and Chase grew viruses (T2 bacteriophage) in one of two isotopic mediums
in order to radioactively label a specific viral component
○
Viruses grown in radioactive sulfur (35S) had radiolabeled 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 people while the smaller viruses
remained in the supernatant.
● The bacterial pellet was found to be radioactive when infected b y the
32P-viruses (DNA) but not the 35S-viruses
● This demonstrated that DNA, not protein was the genetic material, because
DNA was transferred to the bacteria.
Through the X-Ray diffraction, Rosalind Franklin discovered that DNA is a
double helix.
■ 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 with it
contacted an atom
■ The scattering pattern of the X-ray was recorded on a film and used to
elucidate the 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 from an outer backbone
● Shape: The DNA molecule twists at regular
intervals (every 34 Angstrom) to form a
helix (two strands= double helix)
Now that the scientists know that DNA contains the genetic material and that it is
a double helix, they
still need to know how these 2 strands attach.
Chargaff analyzed the composition of DNA and discovered the principle of base
pairing. He did that by discovering that the number of purine bases, which are
double-ring bases (adenine and guanine) always equalled the number of pyrimidines,
which are single-rings bases (cytosine and thymine) and that the number of adenine
equals the number of thymine and the number of cytosine equals the number of
guanine.
DNA replication:
DNA sequencing:
Sequencing is a technique by which the nucleotide base order of a DNA sequence is
elucidated (typically via Sanger method)
● Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group needed to form
covalent bonds (they terminate replication)
● Four PCR mixtures are prepared – each with stocks of normal bases and one
dideoxynucleotide (ddA, ddT, ddG, ddC) Whenever the dideoxynucleotide is
randomly incorporated, the DNA sequence is terminated at that base position
● Because a complete PCR cycle generates millions of sequences, every base position
is likely to have been terminated These sequences are separated by gel
electrophoresis to determine base sequence (according to ascending sequence
length) Automated machines can determine the sequence quickly if fluorescent
labeling of the dideoxynucleotides has occurred
DNA replication:
DNA replication is a semi-conservative process that is carried out by a complex system
of enzymes:
Helicase:
It unwinds and The double-stranded DNA by breaking the hydrogen bonds between
base pairs this occurs at specific regions creating a replication fork of two strands
running antiparallel directions
DNA Gyrase reduces supercoiling by relaxing tension which builds up during DNA
unwinding, preventing DNA breakage.
SSB single stranded binding proteins:
These proteins bind to the DNA atrands after they have been separated and prevent the
strand from re-annealing.
And prevent the single strandeed DNA from being digested by nucleases.
DNA primase: It generates a small RNA primer on each of the template strands
And it proves ann initiation point for DNA polymerase III which can extend a nucleotide
chain but cannot start one.
DNA polymerase III:
DNA polymerase III attaches to the 3’ end of the primer and covalently joins the free
nucleotides (a attaches to T and C with G) together in 5’ to 3’ direction
And as DNA strands are antiparallel, DNA polymerase moves in opposite directions on
the two strands. On the leading stand, it moves towards the replication fork and can
synthesise continuously. On the lagging strand, DNA polymerase iii is moving away
from the replication fork and synthesizes in pieces that are called okazaki fragments.
DNA polymerase I removes the RNA primers from the lagging strand and replaces them
with nucleotides.
DNA ligase joins the okazaki fragments on the lagging strand to form a continuous
strand. And it does this by covalently joining the sugar-phosphate backbones together
with a phosphodiester bond