Molecular mechanisms of
mutagenesis, spontaneous and
induced mutations, and
reversions
1
Mutation - is a change of the nucleotide
sequence of the genome of an organism, virus, or
extra chromosomal genetic element.
- process
Mutations = the source of genetic variability
• heritable changes in the genetic material:
- changes at the level of individual
genes (substitutions, insertions, deletions)
- changes in the structure or number
of chromosomes
not to be mistaken with changes in genotype
(and hence the phenotype), which are the
result of new combinations of existing
genetic variants (recombination)
source of genetic variability - providing new
genetic variants that are a prerequisite for
the evolution of organisms
2
Occurrence and distribution of mutations
• in unicellular organisms, each mutation duplicates in
replication and passes to the next cell generation
• in multicellular organisms, mutations are
transmitted to offspring only when they appear in
the genome of cells of the germ line
• mutations in the DNA of somatic cells occur only in
the progeny of these cells (population of cells that
are genetically different from the rest of the
body)
• Classification of mutations:
• gametic: only in cells the germ line, can cause
hereditary diseases
• somatic: only in somatic cells, may cause cancer
3
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Diploid state protects the cells against
the harmful effects of mutations
• diploid organisms: each gene has two copies
• when one becomes damaged, the other to provides the
correct gene / protein - recessive mutations
• preventing the defect (if the mutation is not dominant)
• estimation: each person carries many harmful
mutations that would in haploid configuration mean
eightfold lethality
• due to the accumulation of mutations over the
centuries, today's people are genetically different from
their predecessors
5
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Range of
mutations? What
do the mutations
influence?
• affect the structure of genes (and their products) or
regulatory regions of DNA
• changing the structure of chromosomes (cause
chromosome aberrations: duplications, deletions,
inversions, translocations)
• the structure of the genome (chromosome numbers
vary:
• aneuploidy - change in the number of certain
chromosomes - monosomy, trisomy - eg. Down syndrome
• euploidy - change in chromosome set number - haploidy,
polyploidy) 7
8
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Are mutations are useful or harmful?
• often harmful because it adversely affects the
function of the gene product and thereby damaging
the cells
• damaged new product may not be only protein but
also RNA (tRNA, rRNA, etc.)
• mutations can also damage the non-coding, but
important signal sequences
• most mutations have no significant impact on the
survival of the organism - they are neutral
• rarely mutation has a positive effect on the survival
and reproduction of the organism
• accumulation of these beneficial mutations will
enable the development of an organism in a changing
environment
10
Expression of Wild-type and Mutant Alleles
11
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• Sickle-cell anemia - hemoglobin S
• Mutation in alleles HBBA change that led to the emergence of alleles HBSS, the
substitution of nucleotide pair T: A to A: T, with thymine in rewriting the chain in the
first case and the second case adenine
• Phenylketonuria
The best studied hereditary disease in phenylalanine-tyrosine
metabolism is phenylketonuria, autosomal recessive disease which is
caused by the lack of phenylalanine hydroxylase enzyme which
converts phenylalanine to tyrosine. Newborns affected by
phenylketonuria should have a strict diet minimal in phenylalanine,
otherwise they may develop severe mental retardation
• Albinism
Albinism, a disorder caused by a lack of pigmentation of the skin, hair
and eyes, is the result of mutations blocking the conversion of
tyrosine to dark pigment melanin
13
Types of mutations
• point mutation: substitution of a single base
• null mutation: complete loss of gene function
• tight mutation has a clear phenotype (eg. full
loss of ability to grow under certain conditions or prevent formation of
product of the biochemical pathway, if the mutation knocks out
an enzyme)
• leaky mutation: partial activity of the gene product is
maintained (eg. the residual activity of the enzyme allows at least slow
growth under certain circumstances)
• direct mutation - standard allele changes in mutant
• back mutation (reversion) - mutant allele changes into standard
• suppressor mutation - a second mutation, which offsets the effect of the
first direct mutation
14
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• Mutation: A Reversible
Process Forward
mutation—mutation of a
wild- type allele to a mutant
allele. Reverse mutation
(reversion)—a second
mutation that restores the
original phenotype. –Back
mutation—a second mutation
at the same site. –
Suppressor mutation—a
second mutation at a
different location in the
genome.
Types of mutations
• substitution: change in the sequence of bases
• inversion: a piece of DNA is inverted, remains in the same place
• duplication: a piece of DNA is duplicated, the second copy usually
remains in the same place as the original
• deletion: one or more bases are removed
• insertion: one or more bases are added
• translocation: stretch of DNA is transferred from its original location
to another location - either the same or different DNA molecule16
Base substitution
• transition: pyrimidine
is replaced by another
pyrimidine (T for C
and vice versa) or the
purine is replaced by
another purine (A for
G and vice versa)
• transversion:
pyrimidine is replaced
by a purine or vice
versa
17
Consequences of substitution
• missense mutation:
TGG (Trp) -> CGG (Arg)
• nonsense mutation – causes formation of stop
codon:
TGG (Trp) -> TAG (Term.)
• neutral mutations - changes and are not reflected in
the function:
AAA (Lys) -> AGA (Arg)
• silent mutation - a different codon, but the same AA:
AAA (Lys) -> AAG (Lys)
18
Severity of mutation depends on their type and
location
• missense mutations are the most common - lead to the
replacement of one amino acid in a protein for another
• when the original amino acid mutations is replaced by chemically
related - usually not serious consequences (conservative
substitution)
• the consequences are serious when changes folding or structure of
the protein active site (radical replacement)
19
Nonsense mutations
• mutations, when the codons encoding amino acids
arechanged to meaningless codons - terminator (UAA,
UAG, UGA in RNA)
• cause premature termination of the synthesis of
polypeptide chain
• truncated polypeptide is not folded correctly
• usually undergoes degradation
20
Transactivation Sequence-specific DNA binding
3´- 5´ Exonuclease
Oligomerization
50 150 200 250 300 350 393100
N CI II III IV V
NLS
C-terminal DNA binding
PuPuPuC(A/T)(T/A)GPyPyPy
TP53 point mutations will cause loss of p53 function as a tumor
suppressor
R175
G245
R249
R248
R273
R282
175
245
248
273
282
Cho et al. 1994
Different reactions of normal and tumor cells to the
oncogenic stress
wtp53 tumor suppressor
mutant p53
A.Merlo, 2004
Insertions and deletions
• Insertion or loss of DNA section
• incorporation into the DNA segment into coding sequence will
usually cause its inactivation
• effect depends on the extent and location (shorter insertion
may allow at least partial activity of the original protein)
• mobile genetic elements (transposons) can be as long as
several thousand bp
23
Insertion can activate gene expression
• change of
repressor
binding site
• change in
promoter - e.g.
conversion of
gene regulation
under the
control of the
transposon
promoter
24
Mutations changing the reading frame
• bases are read as codons (3 bases)
• the inclusion or removal of one or two bases fundamentally changes the genetic
information - leading to loss of protein function
• insertion or deletion of three bases - reading frame does not change - the
protein has 1 AA more or less, its function is usually not significantly altered
25
Inversion
• reversal of a DNA segment
• interruption of the coding sequence - loss of gene function
• when terminal sequences of the inverted portion are in
intergenic regions (inversion includes promoter) - gene
remains intact, even if it is transcribed in the reverse
orientation - does not lose its function
26
Translocation
• exclusion of a DNA segment from its original site and its
insertion at the same or another chromosome
• if the coding sequence is intact - protein function may not
be lost
• inclusion of one gene to another gene - loss or change in
function
27
Duplication
• stretch of DNA is duplicated and both copies remain on
the chromosome
• usually in the location of the original and the copy
adjacent to it (tandem duplication)
• can generate two copies of gene - subsequent divergence
allows the creation of new genes during evolution
• multiple duplication (amplification) may significantly
increase the gene copy number and thus the level of
product
28
Silent mutations
• does not alter the phenotype
• in the noncoding intergenic regions
• introns (can not be in critical splicing sites)
• mutations changing codon sense (e.g. glutamic acid
codons : GAA, GAG, for alanine 4 codons exist: GCU, GCC,
GCA, GCG)
29
Reversion = back mutation
reversion = complete recovery of sequence
pseudoreversion = codon restoring the original function of the
polypeptide
intragenic suppressor mutations = restores the original phenotype
after suppressor-sensitive mutation in the same gene occurs
intergenic suppressor mutations = arises in another gene. Suppressor
is a mutant allele of the gene - suppressing phenotype of suppressorsensitive
mutation
30
Alleles and phenotype
Allele = specific form of a gene
Standard allele = prevalent in the population
Mutant allele = changed by mutation
standard phenotype = standard allele in the
phenotype
mutant phenotype = expression of the mutant
allele in phenotype
31
KEY POINTS
• Mutations occur in both embryonic and the somatic
cells but to the progeny are transmitted only mutations
in the germ cells.
• Mutations can arise spontaneously or may be induced
by mutagenic substances in the environment.
• The mutation is usually a non-adaptive process in
which the individulals with preexisting randomly arising
mutations are selected in given conditions.
• Restoring the standard phenotype in the mutant
organism is the result of back or suppressor mutations.
32
Spontaneous and induced
mutations
Spontaneous mutations occur without apparent
external cause – result of metabolic disorders in the
body, mistakes in DNA replication or the presence
of unknown substances in the environment
induced mutations are formed by the known
physical, chemical or biological factors capable of
inducing changes in DNA - mutagens
Mutagens are also causes of neoplastic
transformation - carcinogens
1. mutagenic directly
2. promutagens, metabolic activation
33
Spontaneous
mutations
Incorrect base pairing
tautomeric base changes
wobble base pairing
depurination and depyrimidination of bases
deamination of bases
uracil incorporation into DNA during replication
oxidative DNA damage
34
Molecular basis of mutation
• structures of bases in
DNA are not static.
Hydrogen atoms can
move from one
position in purine or
pyrimidine to another
position - for
example, from the
amino group to the
ring.
• Such chemical
modifications are
referred to as
tautomerism.
Although tautomeric
rearrangements are
rare, they can have an
important role in
metabolism of DNA,
because some of them
are changing the base
pairing
35
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Tautomeric Shifts Affect Base-Pairing
Mutation Caused by Tautomeric Shifts
37
Wobble base pairing
between tRNA anticodon and the mRNA codon
during replication
correction mechanism of DNA polymerase III
38
Incorporation of uracil into DNA
 associated with spontaneous deamination of cytosine and producing uracil
 occasionally incorporated into the DNA - removed by uracil-DNA-
glycosylase
 a frequent phenomenon in human lymphocytes
 transversion causes GC to AT
39
Oxidative DNA damage
produces hydroxyl radical OH
formed from the hydrogen peroxide in respiratory
chain
causes transversion of GC to AT
40
Induced mutations:chemical mutagens
• influencing the structure of DNA
• oxidizing agents (peroxides, oxygen radicals)
• deaminating substances (nitrites)
• alkylating agents (ethyl methanesulfonate, yperite)
• intercalating agents (acridines)
• aromatic amines (benzidine, naphthylamine)
• substances damaging cellular machinery for the equitable
division of genetic information during cell division –
colchicine
• substances inducing mutation, regardless of the ongoing
replication of DNA (e.g., alkylating agents, nitrous acid)
• substances causing mutations only in DNA replication
(base analogues, acridine dye)
41
Mutagenic base analogues
• structurally similar to the normal bases
• incorporated into the DNA during replication
• structural differences from the normal bases, increase
the rate of mismatch thereby creating mutations
• e.g. 5-bromouracil: thymine analogue - induces a
transition from AT to GC (different charge distribution
- increased frequency of tautomerization to enol form,
which is paired with guanine)
42
Nitrous acid
• causing the oxidative deamination of amino groups of
adenine, guanine and cytosine: amino groups thereby
changing to keto group
• adenine deamination - changed to hypoxanthine, which is
paired with cytosine
• cytosine is deaminated to uracil, which pairs with adenine
43
Alkylating agents
• Ethyl methanesulfonate, aziridine, yperite
• transfer of a methyl or ethyl groups to DNA bases, which
causes a change in base-pairing
• induces all types of mutations (transitions, transversions,
frameshift mutations and chromosomal aberrations)
44
Acridine dyes
• e.g. proflavine, acridine orange, acridine blue
• intercalators – incorporation to DNA groove
• tighten and alter the conformation of the DNA double helix
• during replication occur deletions or insertions of one or
more base pairs - often frameshift mutation (change of
reading frame)
45
Hydroxylamine
• NH2OH
• causes hydroxylation of amino group of cytosine
• resulting hydroxylaminecytosine pairs with adenine
(transition of GC to AT)
46
Physical mutagens
• ionizing radiation (X-ray, gamma, cosmic) induces breaks in
DNA
• non-ionizing radiation (UV) absorption at a specific
wavelength of 260-280 nm, formation of thymine dimers
• the degree of DNA damage is equivalent to the type and
dose of radiation absorbed
47
Mutations induced by ionizing radiation
• shorter wavelength and higher energy than the
visible light
• penetrates deep into the tissue, strikes the
atoms, releases electrons to form positively
charged ions and radicals which give rise to other
ions (ionization process)
48
UV radiation
• less energy than ionizing radiation
• only penetrates into the upper layers of cells, potent
mutagen in unicellular organisms
• not causing ionization
• radiant energy is captured by atoms, the electrons pass
into the excited state - to increase the reactivity of
atoms and molecules in DNA that leads to creation of
mutations
• most mutagenic effects at 254 nm (absorption maximum
of the bases at this wavelength)
49
UV and pyrimidines
• After UV absorption pyrimidines react to
form pyrimidine dimers and hydrates
• thymine dimers disrupt the structure of DNA
and disrupt the replication
50
Biological mutagens
• Viruses - incorporation
into the DNA of of the
host
• transposable elementstransposons:
DNA regions
which can be be moved
from one place to another
in the genome
• Insertion of transposon
may inactivate the gene
(mutagenic)
51
2. DNA repair mechanisms
52
Living organisms contain many
proteins that scan their DNA
for damage and initiate repair
processes when damage is
detected.
DNA repair
• In cells, there are mechanisms by which cell recognizes and completely or
to some extent removes DNA damage. These repair mechanisms are
catalyzed by different sets of enzymes.
• Ability to repair damaged DNA is essential for maintaining the integrity of
the genome of the cell and for the normal functioning of a multicellular
organism.
• Tomas R. Lindahl, Paul L. Modrich, Aziz Sancar won the 2015 Nobel Prize in
Chemistry for his research on the molecular mechanisms of DNA repair.
53
Cell cycle arrest senescence
Inhibition of transcription, replication,
segregation of chromosomes apoptosis
Mutation, chromosome aberration
cancer, heriditary diseases
Types of DNA repair:
• complete repair - repairs to the original
state without DNA synthesis
• excision repair - excision of damaged sites,
synthesis of DNA
• tolerant repair - restoration of function
without complete DNA damage repair
54
DNA repair mechanisms
• enzymes seek DNA damage and when it is found,
they activate any of the repair processes
existing from bacteria to humans
• mismatch repair controlled by methylation
• excision repair (base and nucleotide)
• photoreactivation-correction dependent on the
light - only in bacteria
• postreplication repair
• error-prone repair (SOS response)
• Mutation frequencies: 10-10 mutations / bp /
replication
55
E.coli: 5 mechanisms (photoreactivation, excision repair,
mismatch repair, postreplication, error-prone)
mammals: all except for photoreactivation
1. Photoreactivation
• only in bacteria, eukaryotes, but
not in mammals
• complete repair
• correction dependent on light
• removes thymine dimers mediated
by the enzyme DNA photolyase,
which is activated by visible light
(especially blue 340-400nm)
• DNA photolyase recognizes dimers,
binds to them and cleaves covalent
cross-linking using light energy
• binding to dimers occurs in the
dark, cleavage only after activation
by light energy
• No endonuclease, no polymerase, no
DNA ligase
56
2. Excision repair
(Excision of the damaged sites, synthesis of new DNA)
• performed in three steps:
• DNA endonuclease recognizes damaged base in DNA,
it binds to it and cleaves it
• DNA polymerase fills the gap using the intact
complementary strand as a template
• DNA ligase connects strands retained by DNA
polymerase
• Base excision repair (BER) removes abnormal or
chemically modified bases from DNA
• Nucleotide excision repair (NER) removes larger
defects in DNA
57
2A Base excision repair
• repair of damaged bases (oxidation,
alkylation, deamination), removal of U
• DNA glycosylase creates apurine or
apyrimidine sites (AP sites)
• AP endonucleases recognize AP-space,
which together with phosphodiesterases
cleave - interrupt the sugar-phosphate
backbone, creating 3 OH
• in humans APEX1, APEX2
• DNA polymerase replaces a missing
nucleotide by complementary strand
• Polb in eukaryotes, Pol1 in prokaryotes
• DNA ligase restores the sugar-phosphate
backbone
• Increased risk of colorectal tumors with
mutations in the Polb, DNA glycosylase
58
2B Nucleotide excision
repair (NER)
• removes DNA from
more damaged sites
that distort the
double helix, DNA
adducts, UV
photoproducts
• specific
endonucleases
(excision nuclease)
cleave on both sides
of the section of
damaged nucleotides
• cleavage of the
oligonucleotide
fragment containing
the damaged bases
• gap is filled by DNA
polymerase and
DNA ligase
59
The stability of genes
depends on DNA repair
• repair mechanisms based on the existence of two
copies of the genetic information in the DNA double
helix
• corrupted strand is corrected according to
undamaged
• corrupted strand is identified by abnormal DNA
structures that are generated by errors
• mutations in genes encoding repair proteins
increases the frequency of mutations which
frequently lead to a predisposition to cancer
60
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Inherited DNA repair gene mutations that increase cancer risk
DNA repair gene Protein Repair pathways affected Cancers with increased risk
breast cancer 1 & 2 BRCA1 BRCA2
HRR of double strand breaks and daughter strand
gaps[16] breast, ovarian [17]
ataxia telangiectasia mutated ATM
Different mutations in ATM reduce HRR, SSA or NHEJ
[18] leukemia, lymphoma, breast [18][19]
Nijmegen breakage syndrome NBS (NBN) NHEJ [20] lymphoid cancers [20]
MRE11A MRE11 HRR and NHEJ [21] breast [22]
Bloom syndrome BLM (helicase) HRR [23] leukemia, lymphoma, colon, breast, skin, lung, auditory canal,
tongue, esophagus, stomach, tonsil, larynx, uterus [24]
WRN WRN HRR, NHEJ, long patch BER [25] soft tissue sarcoma, colorectal, skin, thyroid, pancreas [26]
RECQL4 RECQ4 Helicase likely active in HRR [27] basal cell carcinoma, squamous cell carcinoma, intraepidermal
carcinoma [28]
Fanconi anemia genes
FANCA,B,C,D1,D2,E,F,G,I,J,L,M,N
FANCA etc. HRR and TLS [29] leukemia, liver tumors, solid tumors many areas [30]
XPC, XPE (DDB2) XPC, XPE
Global genomic NER, repairs damage in both
transcribed and untranscribed DNA [31][32] skin cancer (melanoma and non-melanoma) [31][32]
XPA, XPB, XPD, XPF, XPG
XPA XPB XPD
XPF XPG
Transcription coupled NER repairs the transcribed
strands of transcriptionally active genes [33] skin cancer (melanoma and non-melanoma) [33]
XPV (also called polymerase H) XPV (POLH) Translesion synthesis (TLS) [34] skin cancers (basal cell, squamous cell, melanoma) [34]
mutS H2, mutS homolog 6, mutL
(E. coli) homolog 1, postmeiotic
segregation increased 2 (S.
cerevisiae)
MSH2 MSH6
MLH1 PMS2
MMR [35] colorectal, endometrial [35]
mutY homolog (E. coli) MUTYH BER of A paired with 8-oxo-dG [36] colon [36]
TP53 P53
Direct role in HRR, BER, NER and acts in DNA damage
response[37] for those pathways and for NHEJ and
MMR [38]
sarcomas, breast cancers, brain tumors, and adrenocortical
carcinomas [39]
NTHL1 NTHL1 BER for Tg, FapyG, 5-hC, 5-hU in dsDNA[40] Colon cancer, endometrial cancer, duodenal cancer, basal-cell
carcinoma[41]
The similarity of proteins in
different species
• thanks to the repair mechanisms (and selection
pressure) changes in the DNA structure during
evolution accumulates slowly
• between man and chimpanzee is about 5 million
years divergence, but their nucleotide
sequences in DNA are 98% identical
• Thanks to the precision replication processes
and reparations occurred over millions of years,
only minimal changes in the genetic information
62
Hereditary diseases in humans caused by
disorders in DNA repair
• xeroderma pigmentosum
• Ataxia-telangiectasia
• Fanconi anemia
• Bloom syndrome
• Werner syndrome
• Rothmund-Thompson syndrome
• Nijmegen breakage syndrome
• Errors in DNA repair (damaged excision repair) proteins,
helicases
• high frequency of chromosomal aberrations
• high risk of malignancy
63
Hereditary diseases in humans caused by
disorders in DNA repair
• xeroderma pigmentosum – extreme sensitivity to
sunlight, skin cancer formation
• It affects about 1 in 250,000 newborns
• mutations in genes encoding proteins involved in
nucleotide excision repair – excision nuclease
activity
64
Cells in individuals with XP have a malfunction in the repair of DNA damage
induced by UV radiation, as thymine dimers. XP disease can arise as a
result of defects in any one of at least eight different genes. Products of
seven of these genes, XPA, XPB, XPC, XPD, XPE, XPG and XPF are
required for nucleoside are nucleotide excision repair
Hereditary diseases in humans caused by
disorders in DNA repair
• Cockayne syndrome – short stature, impaired
mental ability
• Trichothiodystrophy - short limbs, brittle hair,
scaly skin, psychomotoric retardation
• also a consequence of disturbances in nucleotide
excision repair
65
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