Mutations & DNA Repair

Assoc. Prof. Dr. Atif Amin Baig ,

Faculty of Medical Sciences,
University Sultan Zainal Abidin.
atifamin@unisza.edu.my
Recalls?

IVF and SCNT

Types
Stem
Cells?
Vitals?
Asymmetrical
cell division
Learning Outcomes

1. What are mutations?

2. How are they induced in-vivo and in-vitro?
3. Types of mutations
4. Quantified Mutations Vs Non Quantified Mutations
5. Level of Mutations
6. Molecular Mutations
7. Role of Polymerase and Endonuclease against mutations?
8. Why we need to repair DNA damage?
9. What are different mechanism by which we can repair DNA?
10. Error Prone and Error free DNA repair systems
What are Mutations?

 Mutation
• --A change in the genetic material (molecular level)

 Mutant
 --an organism that exhibits a novel phenotype

 They act as a source of the Genetic Variability Required for Evolution

Classification of the mutation (In vivo)

1. Spontaneous vs. induced mutation

2. Gametic vs. somatic mutation

3. Lethal or conditional mutation

 Spontaneous mutations

• are those that happen naturally

• no specific agents are associated with their occurence

• and they are generally assumed to be random changes in the

nucleotide sequences of genes
Induced mutations

• those that result from the influence of any artificial factor

• various forms of radiation
• a wide spectrum of chemical agents
• biological agents (e.g. viruses)
Gametic vs. somatic mutations

Mutation arising in somatic cells are not transmitted

to future generations

Mutations in gametes or gamete-forming tissue are

of greater significance because they are transmitted
to offspring as part of the germ line
• dominant
• recessive
• X-linked
Lethal vs. conditional mutations
Mutation may interrupt a process that is essential to the
survival of the organism
- in this case, it is reffered to as a lethal mutation

Conditional mutation is present in the genome of an organism,

but it is expressed and can be detected only under certain
conditions
Types of Mutations (In vitro)
As per need we can induce mutations

Most common are Frame shift based and Point Mutations

Missense and Non-sense mutations

Level of Mutations
• Changes in chromosome number and structure

• Point mutation--changes at specific nucleotide in a gene

(A,T,C,G)

• Insertion mutations--insert fragment of DNA

• Deletion mutations--delete fragment of DNA

DNA Damage Vs DNA Mutations?
Are they same?

Failure of
Mutation DNA Damage
Repair System
The Molecular Basis of Mutation

Mutations alter the nucleotide sequences of genes in several ways,

--the substitution of one base pair for another.

(A for T)

--the deletion (or addition) of one or a few base pairs. ( AT…….GC)

Tautomeric Shifts:
--chemical fluctuations,
--conformation states (stable==========unstable)

Py

A:T
.
C:G

Pu

© John Wiley & Sons, Inc.

Tautomeric Shifts Affect Base-Pairing

C:T
T:G.

© John Wiley & Sons, Inc.

Mutation Caused by Tautomeric Shifts
 Base Substitutions
A transition replaces a pyrimidine with another pyrimidine
or a purine for another purine.
A transversion replaces a pyrimidine with a purine or a
purine with a pyrimidine.

© John Wiley & Sons, Inc.

Frameshift Mutations:
alteration of the open reading frame (ORF)
Mutation Frequency

Frame shift, transition, transversion mutations

are infrequent
• Bacteria and phage: 10–8 to 10–10 per nucleotide pair
per generation
• Eukaryotes: 10–7 to 10–9 per nucleotide pair per
generation 7 to 9
1/10 1/10

Silent mutation: UCU=Ser; UCA, UCC, UCG =

Ser
Ionizing Radiation Causes Changes in
Chromosome Structure

Ionizing radiation breaks chromosomes and can cause

deletions, duplications, inversions, and translocations
Stability of carbon-containing molecules
Mutagenesis by Ultraviolet Irradiation

Hydrolysis of cytosine to a
hydrate may cause mis-
pairing during replication

Cross-linking of adjacent
thymine forms thymidine
dimers, which block DNA
replication and activate
DNA repair mechanisms.
UV-A 320 to 400 nm
UV-B/C <300 nm
Types of Chemical Mutagens

Chemicals that are mutagenic to both replicating and non-

replicating DNA (e.g., alkylating agents and nitrous acid)

Chemicals that are mutagenic only to replicating DNA

(e.g., base analogs and acridine dyes)
Chemical Mutagens
 Alkylating Agents
chemicals that donate alkyl groups to other molecules.

induce transitions, transversions, frameshifts, and chromosome aberrations

(anomaly).

Alkylating agents of bases can change base-pairing properties.

(GC to AT)

can also activate errors during repair processes.

A Base Analog: 5-Bromouracil
--similar structures
--incorporated into DNA
--increase frequency of mis-pairing

© John Wiley & Sons, Inc.

Nitrous Acid Causes Oxidative Deamination of Bases

© John Wiley & Sons, Inc.

 Intercalation of an Acridine Dye Causes
Frameshift Mutations

--(+) charges molecules

--Incorporated into DNA
--DNA is more rigid
--Change conformation
(non-bending)
 Hydroxylamine(NH2OH)

• Hydroxylamine is a hydroxylating (OH) agent.

• Hydroxylamine hydroxylates the amino group of cytosine and

leads to G:C A:T transitions. .
 Mutations Induced by Transposons
(Repeats)
Fragmets/segments of DNA that are capable to shift / translocate/move from one location to another

© John Wiley & Sons, Inc.

Expansion of Trinucleotide Repeats

Simple tandem repeats are repeated

sequence of one to six nucleotide pairs (CGG,
CAG and CTG).

Trinucleotide repeats can increase in copy number and cause inherited

diseases (Fragile X Syndrome, Huntington disease, Spinocerebellar ataxia)
 Summary

• Mutations are induced by

• chemicals,
• ionizing irradiation,
• ultraviolet light, and
• endo(exo)genous transposable genetic elements.

• Point mutations are of three types:

(1) Transitions—purine for purine and pyrimidine for pyrimidine substitutions,
(2) Transversions—purine for pyrimidine and pyrimidine for purine substitutions, and
(3) Frameshift mutations—additions or deletions of one or two nucleotide pairs, which
alter the reading frame of the gene distal to the site of the mutation.

Chromatin remodeling==Epigenetics
Mutation: Basic Features of the Process

Mutations occur in all organisms from viruses to humans.

They can occur spontaneously or be induced by mutagenic

agents.

Mutation is usually a random, non-adaptive process.

Factors Influencing the Rate of
Spontaneous Mutations

• Accuracy of the DNA replication machinery

• Efficiency of the mechanisms for the repair of damaged
DNA
• Degree of exposure to mutagenic agents in the
environment
 Types of DNA Damage
1. Deamination: (C  U and A hypoxanthine)
2. Depurination: purine base (A or G) lost
3. T-T and T-C dimers: bases become cross- linked,
T-T more prominent, caused by UV light (UV-C (<280
nm) and UV-B (280-320 nm)
4. Alkylation: an alkyl group (e.g., CH3) gets added
to bases; chemical induced; some harmless, some
cause mutations by mispairing during replication or
stop polymerase altogether
Types of DNA Damage (cont.)
5. Oxidative damage: guanine oxidizes to 8-oxo-
guanine, also cause SS and DS breaks, very
important for organelles
6. Replication errors: wrong nucleotide (or modified nt)
inserted
7. Double-strand breaks (DSB): induced by ionizing
radiation, transposons, topoisomerases, homing
endonucleases, and mechanical stress on
chromosomes
DNA Repair
Definition

Enzymatic correction
of errors in DNA
structure and
sequence that protects
genetic information
against environmental
damage and
replication errors.
Types of DNA Repair
Based on Errors

3. SOS
2. Repair
1. Direct Excision Error Prone

Repair Repair
Error Free
 Types of DNA Damage Summarised
G A T C

ds DNA Break Mismatch

C-U deamination

ss Break
AP site
Covalent X-linking
Thymidine dimer
Direct Repair
NO NEW DNA IS REQUIRED

Cyclobutane pyrimidine dimer photolyase

Excision Repair
UN DAMAGED DNA STRAND PROVIDE DNA TEMPLATE

• Mechanism for the repair of environmental damage to one

strand of DNA
• Loss of Purines due to thermal fluctuation resulting in
formation of pyrimidine dimers by UV irradiation.
• The site of damage is recognized, excised by an endonuclease
• The corrected sequence is copied from the complementary
strand by a polymerase
• The ends of this correct sequence are joined to the rest of the
strand by a ligase
SOS Repair

The SOS response is a global

response to DNA damage in
which the cell cycle is arrested
and DNA repair and mutagenesis
are induced. The system involves
the RecA protein.
Thank you