#health#medicine#biochemistry#cemistryofnaturalproduct

1. MUTATION AND DNA REPAIR

2. DNA alteration (Mutations)
• Definition:
• Mutation is the change of base sequence of
nucleotides in the genetic code due to
replacement, deletion (removal) or insertion
(addition) of one or more bases resulting in
altered gene product and/or regulation, or a
change in gene copy number, or a structural or
numerical abnormality in chromosomes.

3. Cont----
• Causes:
• Physical (most common) such as: UV, X and 
radiations.
• Chemical carcinogens such as anticancer base
analogs and alkylating agents.
• Environmental pollutants-derived oxidative
free radical such as nitrous acid.
• Genomic instability, errors of DNA replication
and defective repair.

4. Types of DNA mutation or alteration:
• a) Single base alteration (point mutation):
• Deamination, e.g., adenine into hypoxanthine
and cytosine into uracil.
• Depurination or depyrimidination, i.e., removal of
a purine or a pyrimidine base.
• Alkylation of a base (mostly guanine) by covalent
addition of an alkyl radical.
• Insertion or deletion of a nucleotide.
• Base-analog incorporation.

5. Cont----
B) Two bases alteration:
• UV-induced thymine–thymine dimer.
• Cross linkage caused by bifunctional alkylating
agent.
c) Strand breakage: Single-strand breaks are
less harmful than double- strand breaks in the
phosphodiester backbone. It is caused by:

6. Cont----
• Ionizing radiation.
• Radioactive disintegration of incorporated
element.
• Oxidative free radical formation.
d) Cross-linkage:
• Two bases in same strand or opposite strands.
• DNA cross-linkage to histones or other
proteins.

7. A. Point mutations:
Definition and types:
• It is a single base change that can be
Transition mutation: a purine base is changed
to another purine base,e.g., adenine into
guanine or a pyrimidine base to another
pyrimidine base, e.g., thymine into cytosine.
Or, transversion mutation: a purine base is
changed into a pyrimidine base and vice versa.

8. Fate (or effect) of point mutation:
Silent mutation
• the changed base leads to a codon, which
produces the same amino acid.
• In this case, the change lies in the third base
of the codon, which has several alternative
names for same amino acid.

9. Missense mutation
• The change occurred either in first or second
base of the codon producing a different amino
acid.
• The effect of missense mutation on the protein
produced is dependent on the position and
nature of the replacement amino acid. This
protein could be normally functioning, partially
functioning, non-functioning or not produced at
all because of defective RNA processing.

10. Cont----
• Example is Sickle cell anemia or hemoglobin S
with normal two  chains and abnormal two 
chains leading to rapid hemolysis due to
mutant glutamate at position 6 into valine.
• Non-sense mutation, i.e., the altered base
results in a non-sense termination codon. This
leads to pre-mature stopping of protein
synthesis

11. Frame shift mutation
• Shift in the trinucleotide reading frame e.g., in
the BAX gene in ovarian cancer resulting in:
• Truncated protein if a non-sense codon is
developed due to the shift.
• Garbled translation into totally different protein
after the shift point.
• A protein may not be produced at all because
mRNA is degraded.
• Insertion or deletion of three bases produces a
protein with an extra or lacking an amino acid. It
has a moderate effect on the produced protein.

12. Mechanisms of repair of DNA
Damage
• Introduction
• Despite proof reading and mismatch repair
during replication, some mismatch bases do
persist
• In addition DNA can be damaged by mutagens
produced in cells or from the environment.
• If the damage is not repaired mutation can
happen leading to cancer.

13. Repair mechanisms
• 1.Recognition of the distorted part of DNA
• 2. Removal or excision of the damaged region
of the DNA strand by DNA polymerase I
• 3. Filling the gap left by the excision of the
damaged DNA by DNA polymerase I
• 4. Sealing the nick in the strand that has
undergone repair by DNA Ligase

14. Systems of repair of DNA Damage
• 1. Excision repair:
• Used to correct many types of DNA damage
that affects only one strand. It has three
mechanisms as follows,
• A. Nucleotide excision repair of thymine-
thymine (or pyrimidine) dimer
• B. Mismatch Repair:
• C. Base excision repair or correction of
deamination of cytosine to uracil:

15. A. Nucleotide excision repair of
thymine-thymine (or pyrimidine)
dimer:
• Thymine-thymine dimer is covalent linkage of two
adjacent thymine bases in a single strand and
occurs spontaneously, or due to chemical,
radiation, or ultraviolet (UV) light damage to DNA
segment.
• These dimers prevent DNA polymerase from
replicating DNA strand beyond the site of the
dimer.
• There are two ways of correcting such damage:

16. Cont----
• 1. DNA photolyase enzyme is photo-
reactivated by photons of UV or blue spectral
region of light to cleave the dimer into its
original bases.
• 2. UV-specific endonuclease:
• This enzyme recognizes the dimer and makes
a nick in the affected DNA strand 8
nucleotides away from the dimer site to the
5'-side.

17. Cont----
• DNA polymerase I fills the gap with new
nucleotides using the healthy strand as a
template in the 5'3' direction by nick
translation.
• Then, UV-specific endonuclease cut the freed
damaged sequence4 nucleotides from the dimer
site away to the 3'-side. Then, the damaged
piece of DNA diffuses away.
• Then, DNA ligase joins the 3' end of the new
DNA and the 5' end of the original DNA.

18. 5'
3' 5'
3'
5'
3' 5'
3'
3' 5'
3'
5'
3' 5'
3'
5'
3' 5'
3'
5'
3' 5'
3'
UV-specific
endonuclease
DNA polymerase I
DNA polymerase I
DNA polymerase I
DNA ligase
5'

19. B. Mismatch Repair:
• Mismatch, e.g., G-T, is due to copying errors
during replication that may also lead to 1 - 5
bases unpair loops.
• The mismatch is recognized by a group of
proteins called MutS, MutL and MutH that
identify the parent DNA strand by its
methylation at GATC sequences.

20. Cont----
• Once identified the mismatch, they cut the
defective DNA strand at the 3’ side of the
mismatch dimer.
• A special exonuclease (exonuclease 1)
hydrolyzes DNA in 3'5' direction to a few
nucleotides 5' the mismatch and releases free
DNA nucleotides.

21. Cont----
• A new DNA is synthesized to fill the gap by
DNA polymerase III
• DNA ligase joins the 3'-end of the new DNA
and the 5'-end of the DNA ahead of it.
• Defects in these Mut proteins lead to specific
types of hereditary types of cancer

22. 5'
3' 5'
3'
5'
3' 5'
3'
5'
3' 5'
3'
5'
3'
5'
3'
5' 3'
Exonuclease 1
DNA polymerase III
DNA polymerase III
DNA ligase
5'
3' 5'
3'
G
G
MutS
MutL
MutH
G
C
G
C
G
C
T
G
T
5'
3'

23. C. Base excision repair or correction
of deamination of cytosine to uracil:
• Oxidative deamination of cytosine into uracil,
adenine into hypoxanthine and guanine into
xanthine occurs spontaneously, or due to
chemical or radiation damage to a single base.
• Since uracil, hypoxanthine and xanthine are
foreign to DNA; they are recognized and
removed from the DNA strand by specific-
DNA glycosylase

24. Cont----
• AP endonuclease cuts the deoxyribose-
phosphate backbone of the affected strand at 5'
end of the site and removes a few nucleotides.
• Then, DNA polymerase I excise the residual
deoxyribose phosphate unit and inserts cytosine,
as dictated by the presence of guanine on the
undamaged complementary strand.
• Then, the deoxyribose-phosphate backbone is
rejoined by a Ligase.

27. 2. Recombinational double strand
breaks repair:
• The mechanism of repair of double DNA
strand breaks that are due to ionizing
radiation, chemotherapy and oxidative free
radicals.
• Two proteins are involved; Ku and DNA-
dependent protein kinase. Both of them
attach to each end of the double strand break,
activate one another other, unwind the duplex

28. Cont----
• (Ku has helicase activity) and search for the
closest DNA sequence of minimum
complementarity in the opposite strands.
• The extranucleotide tails are degraded into
free nucleotides by exonuclease.
• The gaps are filled by DNA polymerase III and
DNA ligase seals the free ends.

29. Ku
DNA-dependent
protein kinase
DNA ligase
Exonuclease
DNA polymerase III