Section F
DNA Damage, Repair and
Recombination
F1 Mutagenesis
F2 DNA Damage
F3 DNA Repair
F4 Recombination
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
F1 Mutagenesis
•
•
•
•
Mutation
Chemical mutagens
Direct mutagenesis
Indirect mutagenesis
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Mutation-I
• Definition: Mutations are permanent,
heritable alteration in the base sequence
of DNA.
• Classification:
1. Point mutation: A single base change,
includes:
 transition: one purine (or pyrimidine)
is replaced by the other purine (or
pyrimidine);
 transversion: a purine replaces a
pyrimidine.
2. Frame-shift mutation : Insertions or
deletions involve the addition or loss of
one or more bases，and the translated
protein sequence to the C-terminal side
of the mutation is completely changed.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Mutation-II
Phenotypic effects of mutation:
• Silent mutation: If the mutation is
(1) in a non-coding or
nonregulatory piece of DNA;
(2) in the third position of a codon.
• Missense mutation: If the mutation results in an altered
amino acid in a gene product, then it is a missense
mutation whose effect can vary from nothing to death,
depending on the position of the amino acid affected.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Mutation-III
Consequences of mutation:
• Tumorigenesis: Mutations that
affect the processes of cell growth
and cell death can result in
tumorigenesis;
• Genetic polymorphisms: The
accumulation of many silent and
other non-death mutations in
populations produces genetic
polymorphisms.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Chemical mutagens
Most chemical mutagens are carcinogens
• Base analogs: are the normal bases with altered base pairing
properties and can cause direct mutagenesis.
• Nitrous acid: deaminates cytosine to produce uracil, which
base-pairs with adenine and causes G•CA•T.
NH2
N
0
O
HNO2
N
C
HN
De-amination 0
N
U
• Alkylating agents: produce alkylated (methylated) bases.
• Intercalators: generate insertion and deletion mutation.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Direct mutagenesis
• Mechanism: If a base analog is not removed by a DNA repair
mechanism before passage of replication fork, then an incorrect
base will be incorporated. The second replication fixes the
mutation permanently in the DNA.
• For example: thymine analog 5-bromo-uracil (B) base-pairs
with adenine (A) and also with guanine (G). After one round of
replication, thus the B causes A•T  G•C.
AGCTTCCTA
TCGAAGGAT
TB
AGCTBCCTA
TCGAAGGAT
Section F: DNA Damage Repair
AGCTBCCTA
TCGAGGGAT
AGCTBCCTA
TCGAAGGAT
B:A
BG
AGCTCCCTA
TCGAGGGAT
AGCTBCCTA
TCGAAGGAT
B:A
GC
Yang Xu, College of Life Sciences
Indirect mutagenesis
• Definition: Indirect mutagenesis results from:
1. The activation of trans-lesion DNA synthesis (SOS)
2. The suppression of proofreading system,
one or more incorrect bases are incorporated in DNA.
• Mechanism:
1. Most lesions in DNA are repaired by error-free direct reversal
or excision repair mechanisms before passage of a replication
fork.
2. If this is not possible, trans-lesion DNA synthesis may take
place and one or more incorrect bases become incorporated
opposite the lesion, so as to maintain the integrity of the
chromosome.
3. Proofreading may be suppressed during this process.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
F2 DNA Damage
•
•
•
•
•
DNA lesions
Spontaneous lesions
Oxidative damage
Alkylation
Bulky adducts
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
• Definition: A lesion is an alteration of the normal
chemical or physical structure of the DNA.
• Mechanism: Some of the nitrogen and carbon atoms in
the heterocyclic ring systems are chemically quite
reactive. Many exogenous agents, such as chemicals and
radiation, can cause structure changes to these positions
and result in DNA lesions.
• Consequences:
1. DNA mutation: If such a lesion was allowed to remain in
the DNA, a mutation could become fixed in the DNA by
direct or indirect mutagenesis;
2. Cell death: If the chemical change may produce a
physical distortion in the DNA which blocks replication
and/or transcription, causing cell death.
Section F: DNA Damage Repair
T
A
…
DNA lesions
C G
–A:T –
G
C
H
C OH
G
T A
T A
T
A
C
G
A:T…...
DPC
G:C…...
T:A
Yang Xu, College of Life Sciences
Spontaneous lesions
• Mechanism:
1. Deamination: Cytosine deaminates to
produce uracil. Any uracil found in DNA is
removed by an enzyme called uracil DNA
glycosylase and is replaced by cytosine.
2. Depurination: It is another spontaneous
hydrolytic reaction that involves cleavage of
the N-glycosylic bond between N-9 of the
purine bases A and G and C-1' of the deoxyribose sugar and hence loss of purine bases
from the DNA backbone.
3. Depyrimidination: it can also occur with
very low frequency.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Oxidative damage
• Definition: Reactive oxygen species (ROS) such as super-oxide
and hydroxyl radicals produce a variety of lesions, such damage
occurs spontaneously but is increased by some exogenous
agents including  -rays.
• Examples:
NH2
-CH2OH
8 =OH
H2 N
8-Oxoguanine
Section F: DNA Damage Repair
HO
2-Oxoadenine
O
5-hydroxymethyluracil
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Alkylation
• Examples for alkylating agents:
O
CH3–S–O–CH3
O
H2N–C–N
O
•
CH2CH3
N=O
Examples for methylated bases:
CH3
NH2
H2N
CH3
Section F: DNA Damage Repair
CH3
H2 N
Yang Xu, College of Life Sciences
Bulky adducts
• Definition: They are bulky covalent adducts which are formed
by physical or chemical agents. They result in loss of base
pairing ability with the opposite strand.
• Examples:
1. Physical agent: UV light.
C–N
S-N
C=O
2. Chemical agent: Aromatic arylating
C=C
agents
P
C–N
G–NH
S-N
C=O
C=C
P450
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
F3 DNA Repair
• Photo-reactivation
• Alkyltransferase
• Excision repair
• Mismatch repair
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Photo-reactivation
• Definition: In the presence of visible light, the DNA
photolyases (photo-reactivating enzymes) can resolve
cyclo-butane pyrimidine dimers into monomer again,
this process is known as photo-reactivation.
• Mechanism: These enzymes have prosthetic groups
which absorb blue light and transfer the energy to the
cyclo-butane ring which is then cleaved.
Photoreactivation is specific for pyrimidine dimers. It
is a direct reversal of a lesion and is error-free.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Alkyltransferase
Mechanism: Mutagenic protection is afforded by an alkyltransferase,
which removes the alkyl group from the directly mutagenic O6alkylguanine. The alkyl group is transferred to the protein itself
and inactivates it. Thus, each alkyltransferase can only be used
once.
Cys-SH
Cys-SHCH3
CH3
H2 N
H2 N
• Photo-reactivation and alkyl-transferase effect are all belong to
the error-free direct reversal forms.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Nucleotide excision repair (NER)
•
Nucleotide excision repair (NER):
In NER,  an endonuclease
makes nicks on either side of the
lesion,  then the lesion is
removed to leave a gap.  This
gap is then filled by a DNA
polymerase I,  and DNA ligase
makes final phosphodiester bond.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Base excision repair (BER)
• Base excision repair (BER):
 the lesion is removed by a specific
DNA glycosylase, which cleave
the N-glycosylic bond between
the altered base and the sugar.
 This results AP site, then it is
cleaved and expanded to a gap by
an AP endonuclease plus
exonuclease.
 This gap is then filled by a DNA
polymerase I,
 and DNA ligase makes final
phosphodi-ester bond.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Mismatch repair
• Mechanism:
1. Mismatch: In a replicational mispair,
for example G•T or C•A, the wrong
base is only in the daughter strand.
For mismatch repair,  wrong pair
and  wrong strand must be known.
2. Distinguish:  the wrong pair
(mismatch) is identified and attached
by MutS/MutL protein complex, 
and the wrong (daughter) strand is
identified by MutH endonuclease
according to the hemi-methylated
sequence GATC.
3. Excision repair: MutH endonuclease
then makes nicks at GATC site on
either side of the lesion in the newly
synthesized strand, and do excision
repair.
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
F4 Recombination
• Homologous recombination
• Site-specific recombination
• Transposition
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Homologous recombination-I
• Definition: This process involves the exchange of homologous
regions between two DNA molecules, it is also known as postreplication repair. The integrity of DNA containing un-repaired
lesions can be fixed during replication by homologous
recombination.
• Mechanism:
1. In eukaryotes, this commonly occurs during meiosis, when the
homologous duplicated chromosomes line up in parallel in
metaphase I and the non-sister chromatids exchange equivalent
sections by crossing over.
From father
From mother
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
Holliday model
2. In prokaryotes: This model was
proposed by Robin Holliday in 1964.
Mechanism:
 Two homologous DNA align with each
other;
 Nuclease/RecBCD protein complex makes
nicks near the chi sequence
(GCTGGTGG);
 The ssDNA becomes coated in RecA
protein to form RecA-ssDNA filaments.
They cross over and search the opposite
sequence;
 The nicks are sealed and a four-branched
Holliday structure is formed, then branch
migrates;
 The Holliday intermediate can be resolved
into two DNA duplexes in one of two
ways:
Section F: DNA Damage Repair
A
B
a
A
b
B
a
A
b
B
a
b
Yang Xu, College of Life Sciences
Holliday model
Section F: DNA Damage Repair
A
B
a
A
b
B
a
A
b
B
a
b
Yang Xu, College of Life Sciences
Site-specific recombination
• Definition: is mediated by proteins that recognize specific DNA
sequences. It does not require RecA or ssDNA.
• Examples: Prokaryotes: Bacteriophage  DNA / E. coli DNA
attP of λ
p O
p’
B
B’
attB of E.coli
Int (Integrase)
B
Section F: DNA Damage Repair
O
P’
O
Xis (Excisionase)
P
O
B’
Yang Xu, College of Life Sciences
Transposition
Definition: Transposons are small DNA sequences that can move to
almost any position in a cell’s genome. Transposition has also
been called illegitimate recombination because it requires no
homology between sequences nor is it site-specific.
Examples: IS in E. coli
Insertion Sequence:
IS
• They comprise a transposase gene
flanked by ITR.
• The transposase makes a staggered cut
in the chromosomal DNA and then
inserts a copy of IS elements.
• The gaps sealed by DNA polymerase I
and DNA ligase, resulting in a
duplication of the target site.
• The genes between two copies of a
transposon can be deleted by
recombination between them.
Section F: DNA Damage Repair
transposase
--CGTAAGCTGCGGCAGGATTGA-transposase
--GCATTCGACGCCGTCCTAACT---CGTAAGCT
--CGTAAGCTCGCCG
GCGGCAGGATTGA--
--GCATTCGACGCCG
TCCTAACT-CGCCGTCCTAACT--
--CGTAAGCTCGCCG
--CGTAAGCTCGCCGGCGGCAGGATTGA-GCGGCAGGATTGA---GCATTCGACGCCG
--GCATTCGACGCCGCGCCGTCCTAACT-CGCCGTCCTAACT-Yang Xu, College of Life Sciences
Transposition
1983.
Barbara McClintock (86y)
DNA transposable elememt
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences
That’s all for Section F
Section F: DNA Damage Repair
Yang Xu, College of Life Sciences