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DNA Replication, Mutation and Repair
Aims:



1) Mechanism of replication of DNA in eukaryotic cells
2) Discontinuous nature of DNA polymerisation and formation of Okazaki
fragments
3) Proof-reading and error correction by DNA polymerases
4) Ways in which DNA can be damaged by environmental or natural
agents
5) Importance of DNA repair enzymes in controlling mutations
6) Types of mutation: point, insertion and deletion mutations
...

- S Phase à DNA synthesis begins
...

Ø Each new DNA molecule contains one parent and one daughter
strand
...

- The leading strand is synthesised continuously whilst the lagging strand
is synthesised discontinuously (ookazaki fragments)
...
The point where DNA is separated into single strands
and where the new DNA will be synthesised is called the replication fork
...
It
binds loosely to the DNA and is displaced when polymerase enzymes begin
DNA synthesis
...

4) RNA Polymerase Enzyme called PRIMASE copies a short stretch of DNA
strand to create a complementary RNA segment (approx 60 nucleotides
long)
...


What direction
does DNA
polymerase work?

5) There are now 2 single DNA strands available to act as a template
...

DNA polymerase enzymes can only synthesis strands in the 5’ – 3’ direction
(so the template would be 3’ – 5’)
...
DNA polymerase III can now extend the primers
...
Two polymerase enzymes are needed – one for each strand
...

8) The ‘lagging strand’ however has to be synthesised in bits: the
polymerase can only copy a short stretch before it encounters the primer
already sequenced
...

Ø It has to repeatedly release DNA strand and slide further up to
begin extension from another RNA primer
...
These have gaps in between of RNA primers
...

Ø The Okazaki fragments are then joined together by DNA Ligase
(energy requiring process)
...
The exonuclease checks the newly synthesised strand for
incorrect nucleotides and removes them if there is one
...



How is eukaryotic
and prokaryotic
DNA replication
different?




Comment on
mitochondrial DNA
replication?








How many origin of
replication sites do
eukaryotes have in
comparison to
bacteria?









- Mitochondria have their own single circular chromosome (like bacteria)
which is replicated independently from nuclear chromosomes
...

- Eukaryotic cells have multiple replication origins (in bacteria there is
one replication origin per circular bacterial chromosome)
...

- Due to linear chromosomes in eukaryotes, DNA replication is unable to
reach the very end of the chromosomes
...
At the end of a linear chromosome there is no place to
produce the RNA primer to start the last Okazaki fragment
...

- Eukaryotic cells solve problem by:
Ø Having telomere DNA sequences at the end of chromosomes
which are repetitive sequences
...
This enzyme can
extend this repetitive sequence in the 5’ – 3’ direction + DOES NOT
USE CHROMOSOMAL TEMPLATE
...

Ø This prevents it from degrading enzymes
...
This cannot happen in germ cells as the DNA has to be passed
on à BUT as it is a repetitive sequence the telomerase extends the
sequences of telomere region to prevent degradation
...
Somatic cells have no telomerase + so telomere region shortens on
each round of replication until no telomere left + it can no longer replicate –
stoppage of division (senescence) à contributes to the aging process
...

- Cloned animals have shortened telomeres = genetically old?


How do bacteria
solve this problem?

How do eukaryotes
solve this problem?

Do somatic cells
have active
telomerase?

What is the
Hayflick limit?

Why might cloned
animals have a
shorter lifespan?
















What is a point
mutation?







Name the 3 types of
point mutation?




Examples of
diseases?



What is an
insertion or
deletion mutation?

When are these
more catastrophic?


What is cystic
fibrosis caused by?

What causes Cri du
Chat syndrome?

What is an
inversion?

What is a
duplication?

What is a
translocation and
what might it
cause?



Types of Mutation
1) Point mutation – This is where a single nucleotide base has changed
but the reading frame is not altered because it is substituted
...

Ø Missense – Nucleotide base changed and triplet now codes for
a different amino acid
...

Ø Nonsense – Change in nucleotide base which means the amino
acid codon has been altered to a STOP codon resulting in a truncated
protein
...
This may or may not cause a
frame shift
...
Occurs in the loss of 508th codon causing cystic fibrosis if
homozygous
...
People homozygous for loss of 32 CCR5 allele have lost
32nd base pair = HIV resistant
...

Ø May have serious affects on health if in a coding region
...

3) Major deletions – Deletion of large scale or as in Cri du Chat syndrome
deletion of the end of a chromosome (in this case chromosome 5)
...

4) Inversions – This is where a section of the DNA flips over = gross change
in morphology
...
Chronic Myelogenous Leukaemia is caused by pieces of
chromosome 9 and 22 swapping
...

Ø Replication
slippage
–
Sometimes
the
newly
synthesised strand loops out
causing extra nucleotide base(s)
to be added - this is backward
slippage
causing
insertion
mutation or sometimes the
template strand loops out – this
is a forward slippage resulting in
deletion mutation
...

Ø Deamination – This is where an amino group is lost from the
nitrogenous base through hydrolysis
...
Deamination of adenine forms
hypoxanthine which now selectively base pairs with cytosine instead
of thymine (transforms a A-T base pair to a G-C base pair)
...
This could also be the action of
Ultraviolet light (UV-B)
...

Ø Chemical causes – Chemical agents that react with the
nitrogenous bases in nucleotides
...

E
...
superoxide radicals, hydrogen peroxide and hydroxyl
radicals produced by aerobic respiration
...
E
...
Ethidium

What disease does
a defect in the
nucleotide excision
repair proteins
cause?


Bromide (used in electrophoresis as fluorescent marker!)
...


Repairing mutations in DNA



- DNA replication has high fidelity – error rate is low!!
- Nucleotide Excision Repair = A complex of proteins recognises
distortions in the double helix and can detect which strand is newly
synthesised
...
So damaged
region is removed and re-synthesised
...

- Direct Repair = Damage is directly repaired such as through dealkylation
by enzymes
...







Why is HIV so hard
to vaccine against?

What genes are
mutated in cancer
in general?


Detrimental Effects of Mutations
- HIV – This genome has a very high mutation rate due to its reverse
transcriptase (a DNA polymerase) is more error prone than other nucleic
acid polymerase
...

Ø This means it is difficult to find vaccination against it
...

- Cancer – Mutated genes are found in cancer cells and they tend to be
oncogenes and tumour suppressor genes




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