Mutation
Mutation / DNA Damage and
DNA Repair
S.Karthikumar

Effect of Mutation in Human

Mutation
❑A mutation is a change in genetic material.
❑A mutation is a change in the DNA sequence or chromosomal mutation or
arrangement of DNA.
❑Most mutation are the result of error during DNA replication process/ error during
DNA repair. Some types of mutations are known to be caused by certain
chemicals and ionizing radiation, UV.

Mutation Types – Based on Mechanism
❑A Spontaneous
mutation that arises naturally and
not as a result of exposure to
mutagens.
❑Induced mutations caused
by mutagens; Radiation,
Chemicals, or Viruses,…

Spontaneous Mutations
Spontaneous mutations on the molecular level can be caused by :
Tautomerism: A base is changed by the repositioning of a hydrogen atom, altering
the hydrogen bonding pattern of that base, resulting in incorrect base pairing during
replication.
Depurination: Loss of a purine base (A or G) to form an apurinic site (AP site).
Deamination: Hydrolysis changes a normal base to an atypical base containing a
keto group in place of the original amine group. Examples include C → U and A →
HX (hypoxanthine), which can be corrected by DNA repair mechanisms; and 5MeC
(5-methylcytosine) → T, which is less likely to be detected as a mutation because
thymine is a normal DNA base.
Slipped strand mispairing: renaturation in a different spot ("slipping") after
denaturation of the new strand from the template during replication. This can lead to
insertions or deletions.

Induced Mutation : Mutagen
A Mutagen is an agent of substance that can bring about a permanent
alteration to the physical composition of a DNA gene

Mutation Types – Based on Effect on Structure
❑Point mutation
❑Insertion/Deletion

Base substitutions are those mutations in which one base
pair is replaced by another.
❑Transition: The replacement of a base by the other base
of the same chemical category (purine/ purine;
pyrimidine/ pyrimidine).
❑Transversion: The replacement of a base of one chemical
category by a base of the other (pyrimidine/ purine;
purine/ pyrimidine).

The genetic code: how do nucleotides specify 20 amino acids?
• 4 different nucleotides (A, G, C, U)
• Possible codes:
• 1 letter code  4 AAs <20
• 2 letter code  4 x 4 = 16 AAs <20
• 3 letter code  4 x 4 x 4 = 64 AAs >>20
• Three letter code with 64 possibilities for 20 amino acids suggests
that the
• genetic code is degenerate (i.e., more than one codon specifies the
same amino acid).

UAG (Amber),
UAA (Ochre),
UGA (Opal)

Effect of Point Mutation
Silent mutation: Single substitution
mutation when the change in the DNA
base sequence results in a new codon still
coding for the same amino acid.
Missense mutation: One triplet codon
altered, results in one wrong codon and
one wrong amino acid.
Nonsense mutation: Change a codon
that specifies an amino acid into a
termination codon lead to shortened
protein because translation of the mRNA
terminate prematurely.

Sickle Cell Disease
It results from a single base change in the gene
for B-globin. The altered base cause insertion of
the wrong amino acid into one position of B
globin protein.
These altered protein results in distortion of red
blood cells under low oxygen conditions

Insertion/Deletion

Effect of Insertion / Deletion
Insertion/deletion can disrupt the grouping of the codons, resulting in a completely different translation.
Deletions: Remove information from the gene. A deletion could be as small as a single base or as large as the
gene itself.
Insertions: Occur when extra DNA is added into an existing gene.
Multiple of 3 (codon) / Not multiple of 3
Multiple of 3 (codon) causes loss or gain of codons and subsequently amino acids in translated product.
Not multiple of 3
Altered reading frame or fram-shift, altered amino acid sequence, often premature termination of protein
through generation of termination codon with loss of function/activity.

Germline mutation
❑A heritable change in the DNA.
❑Occurred in a germ cell and incorporated in every cell of the body.
❑Can be transmitted to the next generation.
❑Germline mutations play a key role in inherited genetic diseases.
Somatic mutation
❑Occur in any of the cells of the body except germ cell.
❑Can not transmitted to the next generation.
❑These alterations can (but do not always) cause cancer or other diseases.
Mutation Types – Based on Affected Cells

Gain-of-function mutations
❑Change the gene product such that it gains a new and abnormal function.
❑These mutations usually have dominant phenotypes.
❑Often called a neomorphic mutation.
Loss-of-function mutations
❑ The gene product having less or no function.
❑ These mutations usually have recessive phenotypes. Exceptions are when the organism is haploid, or when the
reduced dosage of a normal gene product is not enough for a normal phenotype (haploinsufficiency).
❑When the allele has a complete loss of function (null allele) it is often called an amorphic mutation.
Lethal mutations are mutations that lead to the death of the organisms that carry the mutations.
A back mutation or reversion is a point mutation that restores the original sequence and hence the original phenotype
Mutation Types – Based on Effect on Function

Chromosomal
Mutation

Variation in Chromosome Number
Variations in the chromosome number (heteroploidy) can be mainly of two types:
❑Euploidy
❑Aneuploidy
Euploidy: Change in whole chromosome sets.
❑Monoploidy
❑Diploidy
❑Polyploidy
Aneuploidy: Changes in part of chromosome sets. An additional or missing
chromosome.
❑Hypoploidy: Monosomy, nullisomy
❑Hyperploidy: Trisomy, Tetrasomy

Chromosomal Variation in Number
Aneuploidy
- the abnormal condition were one or more chromosomes of a normal set of
chromosomes are missing or present in more than their usual number of copies.
Nullisomy
- the loss of both pairs of homologous chromosomes
- nullisomics
- 2N-2
Monosomy -the loss of a single chromosome
-monosomics
-2N-1
Trisomy - the gain of an extra copy of a chromosome;
- trisomics
- 2N+1
Tetrasomic - the gain of an extra pair of homologous chromosomes
- Tetrasomics
- 2N+2

Mutation
Rate

DNA Damage
Mutagens
Agents which causes
Mutations

Induced Mutations – Physical Mutagens
1. Exposure to physical mutagens plays a role in genetic research, where they are used to increase mutation frequencies to
provide mutant organisms for study.
2. Radiation (e.g., X rays and UV) induces mutations.
a. X rays are an example of ionizing radiation, which penetrates tissue and collides with molecules, knocking electrons out of orbits
and creating ions.
i. Ions can break covalent bonds, including those in the DNA sugar-phosphate backbone.
ii. Ionizing radiation is the leading cause of human gross chromosomal mutations.
iii. Ionizing radiation kills cells at high doses, and lower doses produce point mutations.
iv. Ionizing radiation has a cumulative effect. A particular dose of radiation results in the same number of mutations whether it
is received over a short or a long period of time.
b. Ultraviolet (UV) causes photochemical changes in the DNA.
i. UV is not energetic enough to induce ionization.
ii. UV has lower-energy wavelengths than X rays, and so has limited penetrating power.
iii. However, UV in the 254–260 nm range is strongly absorbed by purines and pyrimidines, forming abnormal chemical bonds.
(1) A common effect is dimer formation between adjacent pyrimidines, commonly thymines (designated T^T)
(2) C^C, C^T and T^C dimers also occur, but at lower frequency. Any pyrimidine dimer can cause problems during
DNA replication.
(3) Most pyrimidine dimers are repaired, because they produce a bulge in the DNA helix. If enough are unrepaired, cell
death may result.

Production of thymine dimers by ultraviolet light irradiation

Chemical Mutagens
Chemical mutagens may be naturally occurring, or synthetic. They form different groups based on their
mechanism of action:
a. Base analogs depend upon replication, which incorporates a base with alternate states (tautomers) that
allow it to base pair in alternate ways, depending on its state.
i. Analogs are similar to normal nitrogen bases, and so are incorporated into DNA readily.
ii. Once in the DNA, a shift in the analog’s form will cause incorrect base pairing during replication,
leading to mutation.
iii. 5-bromouradil (5BU) is an example. 5BU has a bromine residue instead of the methyl group of
thymine.
(1) Normally 5BU resembles thymine, pairs with adenine and is incorporated into DNA during
replication.
(2) In its rare state, 5BU pairs only with guanine, resulting in a TA-to-CG transition mutation.
iv. Not all base analogs are mutagens, only those that cause base- pair changes (e.g, AZT is a stable
analog that does not shift).

Base-modifying agents can induce mutations at any stage of the cell cycle. They work by modifying the chemical structure
and properties of the bases. Three types are (Figure 19.13):
i. Deaminating agents remove amino groups. An example is nitrous acid (HNO2 ), which deaminates G, C and A.
(1) HNO2 deaminates guanine to produce xanthine, which has the same base pairing as G. No mutation results.
(2) HNO2 deaminates cytosine to produce uracil, which produces a CG-to-TA transition.
(3) HNO2 deaminates adenine to produce hypoxanthine, which pairs with cytosine, causing an AT-to-GC transition.
ii. Hydroxylating agents include hydroxylamine (NH2OH).
(1) NH2OH specifically modifies C with a hydroxyl group (OH), so that it pairs only with A instead of with G.
(2) NH2OH produces only CG-to-TA transitions, and so revertants do not occur with a second treatment.
(3) NH2OH mutants, however, can be reverted by agents that do cause TA-to-CG transitions (e.g., 5BU and HNO2).
iii. Alkylating agents are a diverse group that add alkyl groups to bases. Usually alkylation occurs at the 6-oxygen of G,
producing O6-alkylguanine.
(1) An example is methylmethane sulfonate (MMS), which methylates G to produce O6-alkyl G.
(2) O6-aIkylG pairs with T rather than C, causing GC-to-AT transitions.

Intercalating agents insert themselves between adjacent bases in dsDNA. They are
generally thin, plate-like hydrophobic molecules.
Ethidium Bromide is the common example of intercalating agent
i. At replication, a template that contains an intercalated agent will cause insertion
of a random extra base.
ii. The base-pair addition is complete after another round of replication, during
which the intercalating agent is lost.
iii. If an intercalating agent inserts into new DNA in place of a normal base, the
next round of replication will result in a deletion mutation.
iv. Point deletions and insertions in ORFs result in frameshift mutations. These
mutations show reversion with a second treatment.

Ames Test Protocol
The Ames test is a simple and inexpensive screen for
potential carcinogens. It assays the reversion rate of
mutant strains of Salmonella typhimurium back to wild-
type.
a. Histidine (his) auxotrophs are tested for reversion in the
presence of the chemical, by plating on media lacking the
amino acid histidine.
b. Liver enzymes (the S9 extract) are mixed with the test
chemical to determine whether the liver’s detoxification
pathways convert it to a mutagenic form.
c. The Ames test is used routinely to screen industrial and
agricultural chemicals, and shows a good, but not perfect,
correlation between mutagens and carcinogens.
The Ames test for assaying the potential mutagenicity of chemicals

DNA Repair
Mechanisms
Light-dependent
repair (photo-
reactivation)
Dark Repair
Excision repair.
Mismatch repair
Error-prone repair
system (SOS
response).
DNA Repair

UV Light-
Dependent Repair:
Photolyase Cleaves
Thymine Dimers.
--No endonuclease
--No Poly
--No ligase
LIGHT REPAIR

Excision Repair (steps)
• A DNA repair endonuclease or endonuclease-containing complex
recognizes, binds to, and excised the damaged base or bases.
• A DNA Polymerase fills in the gap, using the undamaged
complementary strand of DNA as a template.
• DNA ligase seals the break left by DNA polymerase.
DARK REPAIR
Types of Excision Repair
• Base excision repair pathways remove abnormal or chemically
modified bases.
• Nucleotide excision repair pathways remove larger defects, such as
thymine diners.

Base Excision Repair
AP:apyrimidinic site

Nucleotide
Excision
Repair

Mismatch Repair
• During DNA replication mistakes can occur as DNA polymerase copies the two strands.
• The wrong nucleotide can be incorporated into one of the strands causing a mismatch.
• Normally there should be an "A" opposite a "T" and "G" opposite a "C".
• If a "G" is mistakenly paired with a "T", this is a potential mutation.
• Fortunately cells have repair mechanisms.
• In this case repair proteins called PMS2, MLH1, MSH6, and MSH2, help recruit an enzyme
called EXO1 that chops out a segment of the mutant strand.
• Then a DNA polymerase can replace the missing section of the strand with a new section
and the mistake is repaired.

The SOS Repair
• If DNA is heavily damaged by mutagenic agents, the SOS response,
which involves many DNA recombination, DNA repair, and DNA
replication proteins, is activated.
• DNA dependent DNA Polymerase V replicates DNA in damaged
regions, but sequences in damaged regions cannot be replicated
accurately.
• This error-prone system eliminates gaps but increases the frequency of
replication errors (Pol II, IV and V are low-fidelity polymerases)

Inherited Human Diseases
with Defects in DNA Repair
Several inherited human disorders result from
defects in DNA repair pathways.

Xeroderma Pigmentosum (XP)
• Individuals with XP are sensitive to
sunlight (UV light).
• The cells of individuals with XP are
deficient in the repair of UV-
induced damage to DNA.
• Individuals with XP may develop
skin cancer or neurological
abnormalities.

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