1. Distinguish between the terms “mutation”, “DNA repair” and “recombination”.
A mutation is an error in the DNA that’s not repaired before the next replication cycle
and therefore, becomes permenant.
Recombination is a process by which a strand of DNA is first denatured from its
dsDNA state and then reannealed to a different ssDNA strand from the same
organism or homologous gene from another organism.
DNA repair are intrinsic mechanisms used by the body to repair faults in DNA and
2. List and briefly explain three major causes for mutation in DNA.
a) Replication errors: Mistakes made during replication that were not detected and
corrected by pol I or III proofreading mechanisms. (A instead of C) if not corrected,
leads to loss of homologous chromosome. (one will have AT, other CG)
b) Spontaneous changes in DNA:
i. 5000 depurinations/human cell/day. (base is lost. The complementary base is
deleted and a frame shift occurs)
ii. 100 spontaneous deaminations of C into U/human cell/day.
iii. A is converted to hypoxanthine which pairs C not T.
c) External factors: Radiation, changes in temperature, mutagens etc.
A. Alkylating chemicals: adds alkyl group to DNA
B. Reactive oxygen species: base pairs with A and C. Highly mutagenic.
C. Non-ionizing radiation: (UV- 260nm) highly absorbed by bases. Leads to
thymine dimers that cause Xeroderma Pigmentosum
D. Ionizing radiation (X rays and gamma rays) double strand breaks.
E. Base analogues: 5 bromouracil=T and replaces it.
F. Intercalating agents: insert between stacked DNA bases. May cause deletion or
addition of bases and therefore frame shift.
1 in 10 mistakes per genome will not be repaired/cell division. So 3 in humans.
N.B: Depurination is spontaneous hydrolysis of N-glycosyl linkage leading to an
abasic site in DNA. (U is read as T by replication machinery)
3. Explain how errors in DNA replication can lead to mutations.
Failure to repair DNA before the next round of replication will lead to permanent
change in DNA (mutation). This is because the two strands will separate and the new
strands formed will carry the complementary bases to the mutated bases and thus,
the mutation now doesn’t need repair and becomes permenant. 4. Distinguish between the effects of mutations on the somatic and germ cells of
A mutation in a somatic cell is not hereditary (not passed vertically), as somatic cells
do not contribute to gametes, however, the mutation can still be detrimental and can
A mutation in the germ cells or the germ cell precursors can lead to hereditary
diseases as the mutated DNA will form the original template used to make the DNA
in the other cells and will therefore, be passed on.
5. List the various types of DNA repair mechanism (we have mentioned seven).
a) Proof-reading by DNA polymerase (I and III) – 3’ to 5’ exonuclease.
b) Direct reversal of damage
c) Base excision repair
d) Nucleotide excision repair
e) Mismatch repair
f) Recombination repair
g) Error-prone repair
6. Give the detailed description of the base excision (or nucleotide excision) repair
process in bacteria.
Base excision repair: (lesion specific)
Base excision is lesion specific. Glycosylase removes N. AP endonuclease cuts
sugarphosphaste phosphodiester bonds on both sides of damaged base. Exonuclease
remove sugar and phosphate. Polymerase synthesizes replacement DNA. Ligase
Nucleotide excision repair:
non lesion specific, detect distortion, UvrA scan and binds with UvrB at the site, UvrB
melts DNA while A leaves and C joins, UvrC nicks both strands (7 to 5’ and 3 to 3’).
7. Describe the mismatch repair process of bacteria (pay attention at the ways in
which the daughter and parent strand are recognized by repair system).
a. MutS scans DNA for distortion→ binds to mismatch
b. MutL recruited by MutS-mismatch-DNA complex
MutS translocates along DNA until a GATC sequence is reached • requires ATP
• binding of MutS at the 2 locations creates loop in DNA
c. MutSL activates MutH (recognizes loop)
d. MutH (endonuclease): recognizes GATC, binds MutSL
e. MutH nicks unmethylated (daughter) strand of DNA → strand is then progressively
excised from GATC to mismatch in 5’ to 3’ direction (using RecJ or exonuclease III)
or in 3’ to 5’ direction (using exonuclease I). A helicase (UvrD) assists.
New DNA strand is synthesized by DNA polymerase III and sealed by DNA ligase
The repair system recognizes the daughter strand as its not methylated yet. (Dam
methylates the A of GATC, so when the dimer binds to the GATC, it recognizes its
methylation status and therefore, recognizes whether the strand is a parent or
N.B: Methylation also occurs on C of CmC(A/T)GG by Dcm. Methylation of Adenine
regulates replication while that of cytosine regulates transcription)
8. Describe briefly the mechanism of direct reversal of damage in bacteria.
Thymine dimers are corrected by photolyase (Light-dependent activity) that breaks
covalent bonds between thymines; chemical basis still unknown - photoreactivation
9. Distinguish between the two DSB repair mechanisms we talked about in class.
(Double stranded breaks)
Recombination repair Nonhomologous end joining
Accurate Error prone
Replication fork encounters lesion in DNA Both strands are broken at the same
that has not been repaired place
The gap is filled using a template from a Double stranded break with no
homologous single strand undamaged complementary strand
Binding of both broken strands by
making blunt ends. Ku70/80 dimmers
connect strands. DNA dependent protein
kinase (DNA-PK) brings and
phosphorylates protein Artemis. Artemis
has exo-endonuclease to make the ends
blunt. DNAP fill in the gap. Ligase and
WRCC4 join them.
10. What is SOS repair mechanism, when is it used and why is it important? It’s an induced multigene response. It’s a highly error prone repair mechanism where
polymerases insert nucleotides independent of base pairing. This introduces
mutations but still enables complete chromosome replication. They induce point
mutations and are therefore preferred to the other repair mechanisms that lead to
frame shifts. It is used when the other mechanisms fail. This is the last repair
mechanism known and if it fails, the cells commit suicide by apoptosis.
11. Describe the term “non-homologous end joining” and explain how this process
results in the repair of double strand breaks in DNA molecule.
NHEJ is a DSB repair mechanism where the broken ends are directly ligated without
the need for a homologous template. The overhangs are removed and the ends are
directly ligated. This could lead to a frame shift but the chromosome is complete
none the less and can be replicated.
12. What are biological roles of DNA recombination?