7.
1 DNA Structure and Replication
In the mid-20th century, there were
questions about whether proteins or DNA
was genetic material.
It was known the viruses which consisted
of only protein and DNA could pass on
genetic material.
Alfred Hershey and Martha Chase
Experiment
- T2 bacteriophages
- Grown in two isotopic mediums
- Viruses were grown in Sulphur-35
or Phosphorus – 32
- P -> radiolabelled DNA
- S -> radiolabelled protein
- Virus infected E.coli, larger bacteria
formed a solid pellet.
- Pellet contained Phosphorus virus
Rosalind Franklin and Maurice Wilkins
- DNA purified then stretched in a
thin glass tube
- Targeted by x-ray beam diffracting
when in contact with atoms
- From scattering pattern the fact
DNA was double stranded, helix
with phosphosphate on the
outside with twists every 34
angstrom and 10 bases per twist.
Replication in short
Helicase unwinds and separates double
stranded DNA by breaking hydrogen bonds
between base pairs (at replication forks)
DNA gyrase relax positive supercoiling via
negative supercoiling to reduce torsional
strain.
Single Stranded Binding Proteins bind to
each strand and prevent re-annealing. SSB
is dislodged when DNA polymerase
reaches it.
DNA primase generates a RNA primer
which is 10-15 nucleotides long
DNA polymerase III attaches to the 3’ end
of primer and moves in the 5’ to 3’
direction, joining free nucleotides
covalently together
Replication occurs towards replication fork
in leading strand and away from
replication fork in lagging strand.
Lagging strands have multiple RNA
primers, DNA polymerase III creates
Okazaki fragments together
DNA polymerase I removes RNA primers
and replaces it with DNA nucleotides
DNA ligase joins Okazaki fragments
together to form a continuous strand
covalently joined via phosphodiester
bonds
Free nucleotides = deoxynucleotide
triphosphates (three phosphate groups)
- DNA polymerase cleaves 2
phosphates and uses that energy
to form phosphodiester bond
Dideoxynucleosides lack a 3’ hydroxyl
group -> prevents phosphodiester bond
formation -> terminator
Dideoxynucleosides can be used in Sanger
method, 4 PCR mixes are set with
nucleotides + ddNTPs
PCR produces all possible terminating
fragments for each base, gel
electrophoresis reveals sequence based on
order of fragments by length.
Distinct radioactive or fluorescent labelled
primers can be used for automated
sequencing.
Short Tandem Repeats (repeating
elements in the non-coding region)
Short tandem Repeats can be removed
with restriction enzymes and used for
profiling because different individuals
typically have different number of results.
DNA packaged -> histones
8 histones -> nucleosome
Nucleosome = protects DNA and facilitates
mobility
Nucleosomes link -> chromatosomes coil
-> solenoid structures condense -> fibres
form loops which are compressed/folded
around a protein scaffold -> chromatin
Negative charge of DNA associates with
positive charge of Amino Acid, during
condensation tails from adjacent histone
octamers link up and draw the
nucleosomes closer together
Histone octamer= nucleosome