DNA MUTATIONS AND REPAIR

Assoc. Prof. Gertrude Kiwanuka

Dept. Biochemistry
MUST
 DNA MUTATION
• Permanent change in the genetic material
(DNA)
• A heritable change in the genetic material.
• Changes in gametes (germ-line mutations) are
passed on to one's offspring
• Damage to DNA of somatic cells (body cells)
known as somatic mutations can cause cancer.
 Importance of mutation
1) They may have deleterious or (rarely)
advantageous consequences to an organism
(or its descendants)
2) Are important to geneticists
• make a variant (mutant) lacking the ability to
perform a process which we want to study.
• Genetic variants possess mutant alleles of the
genes to be studied
3) Major source of genetic variation which fuels
evolutionary change.
 How does DNA get damaged?
1. Endogenous causes
• Cellular metabolic processes
2. Exogenous causes
• Environmental factors
Endogenous
• Mismatch of DNA bases
• Hydrolysis
• Oxidation
• Alkylation
Exogenous
• Ultraviolet radiation (200-300nm)
• Ionizing radiation (e.g. x-rays, gamma rays)
• Chemical agents (e.g. toxic agents)
 What happens if DNA is not repaired?
• Genomic instability
• Apoptosis
• Senescence (no more cell division)
– Results in a lot of problems in the body and one
may not survive
 DNA or chromosomal rearrangements
• Occurs through the process of recombination
– Random events
– They contribute to genetic diversity and can result in
genetic diseases
• Can arise from:
– chromosome breakage, sometimes coupled with failure
in the repair mechanism
– duplications (extra copies)
– translocations (part of a xmosome is transferred to
another xmosome
– mispaired chromosomes during meiosis e.g. in large
deletions
 Chemical mutagenesis

• A mutagen is a physical or chemical (natural or

human-made) agent which can alter the
structure or sequence of DNA.
A. Chemical mutagens: substances that cause
DNA damage
• They result in mismatches in DNA
• When incorporated into DNA, these chemicals
give rise to DNA lesions
• Cause mutations that fall in two major classes
i. Point mutations
• Transitions
• Transversions
 Chemical mutagens (cont’d)
– Transitions : purine changes to purine or pyrimidine
to pyrimidine
– Transversions: purine changes to pyrimidine or
pyrimidine to purine).
– Consequence of a point mutation is a single amino acid
substitution in the polypeptide chain
ii. Insertion or deletion mutations
• One or more nucleotide pairs are inserted in or deleted
from DNA, respectively
 Point mutations
1. Are generated by altered bases (base analogs)
– Structurally resemble purines and pyrimidines and may be
incorporated into DNA in place of the normal bases during
DNA replication:
– Can result from treatment of an organism with base analogs
– E.g. bromouracil (BU)--artificially created compound
extensively used in research.
• Resembles thymine (has Br atom instead of methyl group) and is
incorporated into DNA
• Because of its tautomeric form, it base pairs with guanine instead
of adenine
• Incorporation of Bromouracil into DNA induces a A.T →GC
transition in the next round
 Point mutations
• Aminopurine --adenine
analog can pair with T
or (less well) with C
when A is in the rare
imino form
– causes A:T to G:C or
– G:C to A:T transitions.

•
 Point mutations…
2. Chemicals which alter structure and pairing
properties of bases
– nitrous acid--formed by digestion of nitrites (preservatives)
in foods.
• Oxidatively deaminates aromatic primary amines; converts C to
U, meC to T, and A to hypoxanthine.
– Alkylating agents e.g dimethyl sulfate, nitrogen mustard,
and ethylnitrosourea.
• They attach alkyl groups to nitrogen or oxygen atoms in bases
• E.g. ethylene oxide is used for the sterilization of surgical
instruments
• They often cause transversion mutations
Ethyl methanesulfonate
 Point mutations
3. Deaminating agents turn
• adenine to hypoxanthine;
• guanine to xanthine and,
• cytosine to uracil
• Hypoxanthine in DNA pairs with C and causes
transitions. (Hypoxanthine:C changed to G:C)

NB: Hypoxanthine is similar to adenine except at carbon 6 where it has C=O

Instead of NH2
 Insertion and deletions
Arise from treatment of DNA with intercalating
agents
• E.g. Acridine orange, proflavin, ethidium bromide
(used in labs as dyes and mutagens)
– All are flat, multiple ring molecules which interact
with bases of DNA and insert between them.
– This insertion causes a "stretching" of the DNA
duplex
– DNA polymerase is "fooled" into inserting an extra
base opposite an intercalated molecule.
– Intercalating agents cause frameshift mutations.
 Causes of spontaneous DNA damage
1. Depurination of guanine or adenine to deoxyribose
through hydrolysis of the N-glycosyl linkage
– Is a common form of spontaneous DNA damage
– It leaves an apurinic site in DNA
– About 103 depurinations per 109 base pairs per day
2. Deamination of cytosine to form uracil
– Consequence if not corrected: a C-G base pair becomes A-
T base pair following DNA replication
3. Methylation of bases by S-adenosylmethionine to
form derivatives like 3-methyl adenine, 7-
methylguanine
 Causes
3. Tautomeric shifts
• Normally T is in keto form and pairs with A; it
can spontaneously shift to the enol form,
which pairs with G
– G is incorporated in the new DNA strand instead
of A (T-G pair)
 B: Physical mutagens

DNA damage by environmental factors

• Include high-energy radiation like X-rays and
ultraviolet light
• UV radiation (200-300 nm) promotes formation
of pyrimidine dimers e.g. Thymidine dimers (T-T
dimers)
– Intrastrand covalent bonds forming a cyclobutyl ring
between C5 and C6 of one T and the same carbons
on the adjacent T
– Interfere with replication and transcription
– Are lethal if unrepaired
 Agents altering DNA stucture (cont’d)
• Ionizing radiations e.g. X- and gamma-rays
– they produce reactive ions (charged atoms or
molecules) when they react with biological
molecules
 DNA Repair...
• How is a cell able to repair the > 104
mutations/day/genome?
• A lot of energy is spent on enzymes that monitor
DNA for mutations
• In most DNA damages, only one strand needs
repair
• DNA repair mechanisms distinguish the
damaged & undamaged strands
• Repair enzymes replace the mutated DNA using
information in the complementary strand
 DNA Repair...
• DNA damage must be
repaired before the S
(synthesis) phase of the
cell cycle
– to prevent irreversible
mutation when DNA is
replicated
• Mutagens are most
mutagenic in the S
phase
 DNA repair…
• Cancer cells due to rapid division are likely to
be in the S phase when mutagenic irradiation
is applied
– This is the basis for using radiation for cancer
treatment
 DNA repair…
• Four Basic steps in DNA repair:
1. Recognize
2. Remove
3. Resynthesize
4. Religate
 Four basic types of repair
Repair system Type of damage E. coli proteins Human genes
Nucleotide excision Pyrimidine dimers ABC exonuclease, DNA pol XPA, XPE, XPB, ERCC4
I, DNA ligase
(XP=Xeroderma
pigmentosum)
Mismatch Nucleotide mismatches MutH, MutL, DNA pol III, Mlh1, pms1, msh2 (MutS)
DNA ligase, exonuclease I

Base excision Removal of uracil, DNA glycosylase, AP Yeast DNA pol γ,

hypoxanthine, alkylated endonuclease, DNA pol I, mammalian pol β, human
bases ligase DNA glycosylase

Direct Chemical reversal of O6-methylguanine DNA O6-methylguanine DNA

methylation or methyltransferase methyltransferase
photoactivation
 1. Nucleotide excision repair
• Mechanism is also called bulky lesion repair
• Important in removal of thymine dimers
• The structure recognized by the repair
enzymes is an abnormality in the DNA
backbone
• Xeroderma pigmentosum
– Is a human genetic disease
– Caused by a defect in DNA repair of T dimers
– Characterized by extreme sensitivity to sunlight
with predisposition to skin cancer
Nucleotide excision
 2.Mismatch repair
• Is coupled to replication
• Causes: Base substitution, small insertions and
deletions
• Damage is repaired by the mismatch repair
system
– The mismatch-recognizing component is located
at the replication fork
– It is located System has exonucleases, DNA
polymerase and DNA ligase
– It binds the new strand (lagging) being formed
• Strand has many nicks
 Mismatch repair…
• Newly synthesized DNA strands are also
unmethylated on adenine residues in the
sequence GATC
• In E. coli, 3 proteins: MutS, Mut, H and MutL
correct the errors
• Human homologues of MutS (hMSH2) and
MutL when mutated can result in hereditary
nonpolyposis colon cancer (HPCC)
 3. Base excision
1. Removal of nucleotides
that have lost the base
moiety as a result of
depurination by the
action of DNA
glycosylases
• The driboseMP is removed
by an endonuclease and
phosphodiesterase
• A single nucleotide gap is
created
• DNA polymerase I replaces
the nucleotides
• DNA ligase seals the nick
 4. Direct reversal of damage

• This mechanism does not involve removal of any

nucleotides
• It is independent of DNA pol I and ligase
• E.g. Methylation of guanine at the O6
• O-methyl-guanine can base pair with thymine
• O-methyl-guanine-DNA methyltransferase
removes the CH3 by transfering it to Cys residue
in the enzyme
NB: Thymidine dimers can be restored to their monomeric
forms by photoreactivation catalyzed by photolyases
 Reading assignment
• What is SOS response in DNA repair?
• Homologous recombination and the repair of
double stranded DNA breaks
– non-homologous end-joining and homology
directed repair systems.