DNA Mutations,

Damage & Repair

Lecture 3

How are mutations caused at the

DNA level?
Spontaneous errors

Tautomerism
Replication errors
Depurination
Insertion of DNA via MGE

Induced errors

Chemical
Chemicals
Pollutants
Physical
Radiation (UV, Gamma)

Transitions & Transversions

Induced errors
Types of Chemical agents
i.
ii.
iii.
iv.

Base analogues
Base modifiers
Intercalating agents
DNA crosslinkers

Base Analogues
Causes inaccurate base pairing
Sometimes used in cancer & anti-viral therapy (Why?)
Eg:
a) Adenine analogue 2 amino purine (2AP)
2AP substitutes A (AT GC transition)
b) Thymine analogue 5 Bromouracil (5BU)
5BU substitutes T (AT GC transition)

Base Analogues
5BU substitutes T (AT GC transition)

2AP substitutes A (AT GC transition)

Base Modifiers

Hydroxylamine (NH2OH)

NH2OH reacts with C (hydroxylation)

Hydroxylated C pairs with A (CG TA transition)

Alkylating agents

Transfer alkyl groups (methyl, ethyl) to bases

Alkylation of guanine results in 06-methylguanine (AlkG).

AlkG pairs with T (GC TA transition)

Nitrogen mustards can also alkylate guanines.

- Eg: N-methyl-N-Nitrosoguanidine (NMNG)

Nitrogen mustard (gas chemical warfare)

Base Modifier

Base Modifier

Intercalating Agents
Cause frameshift mutations, small insertions & deletions

Ethidium bromide

4 ringed molecule

Intercalates (inserts) into DNA helix

Changes distances between bases

Additional bases incorporated

DNA Crosslinkers
Occurs when various exogenous or endogenous agents
react with two different positions in the DNA.
This can either occur in the same strand (intrastrand

crosslink) or in the opposite strands of the DNA (interstrand crosslink)

Mitomycin C & Cis platinum

Insert between strands of helix

Form covalent bonds

Strands cannot separate during replication

Physical Agents

Gamma Radiation

Directly on cellular components and can

cause DNA strand breaks
Indirectly by radiolysis of water which
generates ROS products

UV radiation

Energy absorbed directly by DNA

Causes pyrimidine dimers (T=T)
Transition or transversion mutations
(why?)

Radiation
Microwaves
Radar waves
Television waves
Radio waves

X rays
Gamma rays
10-6
10-4
Wavelength
(nm)

100

102

10-2

100

104

106

Sunlight

200

800

Ultraviolet
254nm

108

Visible
400

Infrared
700

1010

UV Radiation
200

250

UVC
Far UV

300

UVB
Near UV

350

UVA

400 nm

VIS

254 - 260 nm
most active
on DNA

Cyclobutyl pyrimidine dimer is the covalent interaction of two adjacent

pyrimidines in the same polynucleotide chain

DNA Damage & Repair

DNA Damage
DNA is damaged when either the sequence of the
bases is changed or the phosphate backbone of
the DNA molecule is changed.
DNA damage prevents the message in the
sequence to be conveyed to daughter molecules.
Estimated that E. coli acquires 3000-5000 changes
in the DNA per chromosome per cell generation.

Exposure of E. coli to DNA damage

(survival curve)
Log surviving fraction of cells

100

Plateau region very slow decrease

Rapid decrease

Exposure time
(min)

Cellular DNA Repair Responses

Damage reversal

Damage excision

Damage accurately corrected without removing

actual base or cutting the strand
Damage repaired by removing damaged base (or
region) and using opposite strand to resynthesise

Damage tolerance

Damage tolerated during replication and repaired

afterwards.

Cellular DNA Repair Responses

I. Damage Reversal (Direct Repair)
Enzymatic Photoreactivation (Light repair)
Methyltransferases

Enzymatyic Photoreactivation

Light dependent process

Enzyme catalysed repair of cyclobutyl pyrimidine dimers

monomerisation of pyrimidine dimers

Wavelengths (320 500nm)

Enzyme = DNA photolyase

light harvesting properties

Chromophore structure increases absorption area

Flavin adenine dinucleotide (FAD) photoexcited

Enzymatyic Photoreactivation

Gene = phr

Occurs plants and animals

eg algae, bacteria, birds, reptiles

Not yet found in mammals.

Enzymatyic Photoreactivation

Mechanism of Enzymatic Photoreactivation

Photolyase scans DNA molecule for pyrimidine dimers and
binds to dimer close to catalytic site together with redox
co-factor FADH. Dimer is flipped out from the DNA.
The FADH in the Photolyase/dimer complex undergoes
photoexcitation (energy generated from absorption of
quantum of light)
This causes electron from FADH to be transferred to the dimer
which breaks the covalent bond between the dimers and
it splits.
An electron returned back to FADH and the photolyase is

released from the DNA.

Pyrimindine Dimer removal in plant tissue

Kimura S et al. Nucl. Acids Res. 2004;32:2760-2767

Photoreactivation is the major DNA repair pathway for UVinduced damage in

nonproliferating cells (leaves) in the presence of sunlight

Methyltransferases
Alkylation of guanine and thymine gives rise to 06methylguanine and 06-methylthymine respectively.

06-methylguanine and 06-methylthymine can bind to

thymine and guanine respectively.
Methyltransferases can transfer methyl group from alkylated
base to itself and allow bases to resume normal pairing.
Once methyltransferases accepts alkyl group it is inactivated
and degraded.

Methyltransferases

Cellular DNA Repair Responses

II. Damage Excision
Base excision Repair (BER)
Nucleotide excision Repair (NER)
Mismatch Repair

Base Excision Repair

DNA Glycosylases: excise bases by hydrolyzing the N, Nglycosidic linkage between the base and the sugar-phosphate
backbone leaving a AP (apurinic or apyrimidinic) site.
Class I
DNA glycosylase WITHOUT AP ENDONUCLEASE activity

3 Methyladenine DNA glycosylase II

alkA gene
General recognition of all alkylated bases
Class II

DNA glycosylase WITH AP ENDONUCLEASE activity

eg: Pyrimidine dimer endonucleases

Base Excision Repair

Base Excision Repair

Step 1
Certain forms of base damage are recognized by DNA glycosylases that
catalyze excision of the damaged base from the sugar-phosphate leaving
an AP (apurinic/apyrymidinic) site in the DNA.
Step 2
AP site recognised by a 5 AP endonuclease which cuts the
phosphodiester backbone on one DNA strand on the 5 side of the AP site
leaving a 5 terminal deoxyribosephosphate moiety.
Step 3 and 4
The gap is filled by DNA polymerase with new DNA and DNA
deoxyribophosphodiesterase (dRpase) catalyzes the excision of the 5
sugar-phosphate moiety.
Step 5
The resulting nucleotide gap following DNA polymerase dissociation is
filled by DNA ligase.