MUNI
SCI
Bi4025en
Molecular Biology
Mgr. Jiří Kohoutek, Ph.D.
1    Department of Experimental Biology
Lecture 8
• Molecular mechanisms of mutagenesis and recombination.
2    Department of Experimental Biology
Source of genetic variability
• Mutations
• Recombination
• Transposition
3    Department of Experimental Biology
MUNI
SCI
Mutations
TABIE 14.1
Major types of mutations and their distinguishing features
Basis of classification	Major types of mutations	Major features
Origin	Spontaneous Induced	Occurs in absence of known mutagen Occurs in presence of known mutagen
Cell type	Somatic Germ-line	Occurs in nonreproductrvc cells Occurs in reproductive cells
Expression	Conditional Unconditional	Expressed only under restrictive conditions (such as high temperature) Expressed under permissive conditions as well as restrictive conditions
Effect on function	Loss-of -function (knockout null) Hypomorphic (leaky) Hyperm orphic Gam of function (ectopic expression)	Eliminates normal function Reduces normal function Increases normal function Expressed at incorrect time or in inappropriate cell types
Molecular change	Nucleotide substitution Transition Transversion Insertion Deletion	One base pair in duplex DNA replaced with a different base pair Pyrimidine (T or C) to pyrimidine, or purine (A or C) to purine Pyrimidine (T or C) to punne, or purine (A or C) to pyrimidine One or more extra nucleotides present One or more missing nucleotides
Effect on translation	Synonymous (silent) Misscnsc (nonsynonymous) Nonsense (termination) Frame-shift	No change in amino acid encoded Change in amino acid encoded Creates translations! termination codon (UAA, UAG. or UGA) Shifts triplet reading of codons out of correct phase
4    Define footer - presentation title / department https://www.uky.edu/~tphillip/2FChapter14.ppt
MUNI
SCI
Mutations
• Mutation is an inherited change in the genetic material of an organism.
• Consequence of replication failure or accidental DNA damage.
• Mostly without significant effect.
• Sometimes fatal.
• Sometimes mutations bring an advantage (e.g. resistance of bacteria to antibiotics).
• Increase diversity among individuals of a given species.
5    Department of Experimental Biology
MUNI
SCI
Mutations
• Mutations occur randomly.
• More likely to cause harm than good.
• Responsible for thousands of human diseases, including cancer.
• The survival of the cell/organism depends on minimizing changes in DNA.
6    Department of Experimental Biology
MUNI
SCI
Mutations
• Change in the primary structure of the nucleic acid:
o at the gene level (base pair substitutions, insertions, deletions), o at the level of structure or number of chromosomes.
• Genotype can also be changed by new combinations of existing genetic variants - alleles, recombination.
• The basis of evolution, adaptation to the environment.
• Use in research (identification of genes, research of their function, regulation of expression, etc.)
7    Department of Experimental Biology
MUNI
SCI
Polymorphism
•A state where there are at least two genetic variants (alleles). Eventually, the occurrence of different forms among the members of a population or colony.
•We talk about a mutation if the percentage of the allele in a population less than 1%.
•Changes in gene expression that are phenotypically manifested, and which are not the result of changes in the DNA nucleotide sequence are referred to as epigenetic.
8    Department of Experimental Biology
MUNI
SCI
Standard x Mutant phenotype
Standard allele - standard phenotype.
Mutant allele - mutant phenotype (mostly recessive).
• Wild type tigers have orange fur and black stripes. One mutation prevents the deposition of the orange/brown pigment, and the result is a "white tiger" that still has dark stripes. A different mutation prevents any melanin (brown pigment) from forming at all, and the result is an albino tiger.
MUNI
SCI
9    Department of Experimental Biology
http://www.bio.miami.edu/dana/dox/wildtype.html
What does mutation affect?
• The sequence of the genes, and thus structure of their products or regulatory areas in DNA.
• Chromosome structure (chromosome aberrations: duplication, deletion, inversion, translocation).
• Genome structure:
• Aneuploidv - change in the number of certain chromosomes.
• Euploidv - change in the number of chromosomal sets.
10 Department of Experimental Biology
MUNI
SCI
Classification of Mutations
• Direction of mutations:
• Forward mutation - mutation changes wild type (ancestral) to mutant (derived).
• „Reverse" mutation - mutation changes mutant (derived) to wild type (ancestral).
o Reversion to the wild type amino acid restores function.
o Reversion to another amino acid partly or fully restores function.
• Suppressor mutation - partially or completely cancels out the effect of another so-called suppressor-sensitive mutation.
o Intragenic suppressors occur on the same codon; e.g., nearby addition
restores a deletion, o Intergenic suppressors occur on a different gene.
11   Department of Experimental Biology
MUNI
SCI
Intragenic suppressor mutations - type I
Intragenic suppressor mutation suppresses suppressor-sensitive mutation at the same gene level (intragen).
suppressor-sensitive mutation
ATC CTC CCT TTC
Insertion of T
ATC TCT CCC TTT C
frameshift mutation (reading frame shift)
12  Department of Experimental Biology
suppressor mutation
i
ATC TCT CCC TTT C
I       Deletion of C ATC TCT CCT TTC
restoration of the original reading frame
MUNI
SCI
Intragenic suppressor mutations - type II
Serine      TCA • Original codon.
| • Suppressor-sensitive (first) mutation.
TAA • Stop codon, loss of product function.
| • Suppressor-sensitive (second) mutation.
Tyrosine    TAT     • Codon for another amino acid, restoration of
function (sometimes only partial).
13 Department of Experimental Biology
MUNI
SCI
Intergenic - Suppressor mutations
• Many function in mRNA translation.
• Each suppressor gene works on only one type of mutation - nonsense, missense, of frameshift mutation.
Suppressor genes often encode tRNAs, which possess anti-codons that recognize stop codons and insert an amino acid.
• Three classes of tRNA nonsense suppressors, one for each stop codon (UAG, UAA, UGA).
o tRNA suppressor genes coexist with wild type tRNAs. o tRNA suppressors compete with release factors, which are important for proper amino acid chain termination.
• Small number of read-through polypeptides are produced; tandem stop codons (UAGUAG) are required to result in correct translation termination.
14 Department of Experimental Biology
MUNI
SCI
Intergenic - Suppressor mutations
• The original mutated gene does not change, but the way of its mRNA translation is affected.
• The actual mutation is suppressor-sensitive (sus-).
• The codon turns into a nonsense codon or missense codon with altered sense.
5' i i i atg
tXc . 3. i i i
tag
translation in the absence of suppressor tRNA
i a t c
mRNA 5 a u g
OH aa
f-Met'
t a a
att
u a a 3'
Met
15 Department of Experimental Biology
MUNI
SCI
Intergenic - Suppressor mutations
• Suppressor mutation: mutation in the tRNA gene, which produces tRNAwith altered antikodon.
• Gene suppressor = mutant allele of genu for tRNA (sup-).
translation in the presence of suppressor tRNA
Department of Experimental Biology
Classification of Mutations
By type of affected cell:
Somatic mutation
Arise in the somatic cells.
Passed on to other cells through the process of
mitosis.
Effect of these mutations depends on the type of the cell in which they occur & the developmental stage of the organism. In single-celled organisms, each mutation is duplicated during replication and passed on to the next cell generation.
In multicellular mutations are passed on to the offspring only if they appear in the genome of germ line cells.
• Germline mutation
• They occur in the cells that produce gametes.
• Can provoke hereditary diseases.
• Passed on to future generations.
• In multicellular organisms, the term mutation is generally used for germ line mutations.
Department of Experimental Biology
MUNI
SCI
Classification of Mutations
• According to the level at which it acts:
• Gene (point) - change of bases or sequence of bases at the gene level.
• Chromosome - change of sequence at the chromosome level.
• Genome - genomic mutations - change in the number of chromosomes.
18 Department of Experimental Biology
MUNI
SCI
Classification of Mutations
• According to the effect on the viability of the organism:
• Vital mutations - compatible with survival.
• Lethal mutations - incompatible with survival.
• Conditionally lethal mutation - compatible with survival under certain conditions
o ts ("temperature-sensitive") - lethal at elevated temperature.
o sus ("suppressor-sensitive") - lethal without the presence of a suppressor.
19 Department of Experimental Biology
MUNI
SCI
Temperature sensitive mutation
A conditional mutation that produces the mutant phenotype in one (restrictive or non-permissive) temperature range and the wild-type phenotype in another (permissive) temperature range.
osxn
Restrictive temperature:
protein not functional
Temperature-sensitive mutant .subunit
Wild-type subunits
High
temperature
Misfolded interfaces block or destabilize assemblies
^ £ 20 seconds ^
Permissive temperature:
protein functional
20 Department of Experimental Biology
A conditional mutation that produces the mutant protein folds correctly only at a lower permissive, temperature.
At non-permissive temperature, the mutant protein is inoperative.
Useful for experimentation (lethal mutations are difficult to study).
MUNI
SCI
Bioessays 36: 836-846, DOI 10.1002/bies.201400062 Methods in Cell Biology, Volume 137, 2017, Pages 283-306.
Classification of Mutations
According to the degree of phenotypic manifestation (in diploid organisms):
Dominant mutations - they manifest themselves even in heterozygous state.
Recessive mutations - they manifest themselves only in a homozygous state, in a heterozygous state, the manifestation is masked by the dominant allele.
Dominant and Recessive mutations
Recessive Dominant
Genotype
üü ÜÜ
V
Phenotype
V /
21   Department of Experimental Biology
MUNI
SCI
Dominant negative mutation
DNA Mtiumi
Transcription and translation
\\'ilil-ti|K' piciu in Muknu prolciii
■ ■
Protein koi.i>in<;
O
.....I
Defective
subunit assembly
• Dominant negative mutations are those where the mutant protein loses its own function but, in addition, the defective protein interferes with the function of another protein.
• Thus a dominant negative mutation usually results from the presence of an altered, defective protein.
• Relate to genes coding for components of multimeric proteins.
• If the protein works as a dimer and in one both standard and mutant proteins appear in the cell non-functional heterodimers are formed.
O O O
3/4 uon-fuiirtionaj
22  Department of Experimental Biology
Possibility of experimental use for targeted inhibition of proteins (research of their function).
https://www.brainkart.com/article/Dominant-Mutations-May-Be-Positive-or-Negative_13969/
MUNI
SCI
Diploid character of genome protects against adverse
effect of mutations
• Diploid cell contains two complete sets of chromosomes, one from each parent.
• In diploid organisms, mammals, each gene has 2 copies.
• If one of them is damaged, the other can substitute the other one by providing the correct information.
• Therefore diploid state prevents defect caused by mutations, only, if the mutation is not dominant.
23 Department of Experimental Biology
MUNI
SCI
Point mutations - molecular change
• 1. Base pair substitutions
• Transitions
o Convert a purine-purine to the other pyrimidine-pyrimidine. o 4 types of transitions; A <-> G and T <-> C o Most transitions results in synonymous substitution because of the degeneracy of the genetic code.
• Transversions
o Convert a purine-pyrimidine to a pyrimidine-purine.
o 8 types of transversions; A <-> T, A <-> C, G <-> T & G <-> C.
o Transversions are more likely to result in nonsynonymous substitution.
2. Base pair substitution, deletions and insertions
24 Department of Experimental Biology
MUNI
SCI
Point mutations molecular change
Cytosine
Guanine
Although there are twice as many possible transversions, because of the molecular mechanisms by which they are generated, transition mutations are generated at higher frequency than transversions.
As well, transitions are less likely to result in amino acid substitutions (due to "wobble"), and are therefore more likely to persist as "silent substitutions" in populations as single nucleotide polymorphisms (SNPs).
25 Department of Experimental Biology
https://www.mun.ca/biology/scarr/Transitions_vs_Transversions.html
MUNI
SCI
Point mutations molecular change
Insertion
Insertions are mutations in which extra base pairs are inserted into a new place in the DNA.
Insertion
C TG G A G
CTGGTGGAG
Deletions
Deletions are mutations in which a section of DNA is lost, or deleted.
Deletion
CTGGAG C T A G
26 Department of Experimental Biology
https://evolution.berkeley.edu/dna-and-mutations/types-of-mutations/
MUNI
SCI
Substitution point mutations
Effect of translation
Substitution mutations change base pairs of a nucleotide sequence with different base pairs.
The substitution may or may not give effects depending on the type of mutation.
Silent mutation, missense mutation and nonsense mutation are three types of substitution mutations.
No mutation
DMA level TTC mRNAIevel AAG pfotein level LyS
Silent
Point mutations
I
Nonsense
ATC UAG STOP
Missense
conservative rwn-conservHlivB
T C AGG Arg
T
TGC AGG Thr
Cast I -.dar
27 Department of Experimental Biology
https://www.differencebetweenxom/difference-between-substitution-insertion-and-deletion-mutations/
MUNI
SCI
Substitution and point mutations
Missense mutation
Base pair substitution results in substitution of a different amino acid.
Nonsense mutation
Base pair substitution results in a stop codon (and shorter polypeptide).
Neutral mutation
Base pair substitution results in substitution of an amino acid with similar chemical properties (protein function is not altered).
Silent mutation
Base pair substitution results in the same amino acid.
Frameshift mutations
Deletions or insertions (not divided by 3) result in translation of incorrect amino acids, stops codons (shorter polypeptides), or read-through of stop codons (longer polypeptides).
MUNI
Department of Experimental Biology r> r» t
Substitution and point mutations
• Missense mutation
• Base pair substitution results in substitution of a different amino acid.
5'	TCTCAA	AATTTACG	3'	5'	TCTCAA	AATT TACG
3'	AGAGTT	TTAAATGC	5'	3'	AGAGTT	TTAAATGC
Ser Gin Lys Phe Thr •■• ••• Ser Gin Phe Thr •
• Nonsense mutation
• Base pair substitution results in a stop codon (and shorter polypeptide).
5'      TCTCAA   AATTTACG      3' 5'      TCTCAA AATTTACG
3'      AGAGTlQTTAAATGC      5' 3'      AG AGTTDT T A AATGC
-•• Ser Gin Lys Phe Thr ■■■ -■■ Ser Gin
Department of Experimental Biology
Substitution and point mutations
Neutral mutation
Base pair substitution results in substitution of an amino acid with similar chemical properties (protein function is not altered).
5' TCTCAAA ATTTACG 3' 3'      AGAGTTT   TAAATGC 5'
5' TCTCAAA ATTTACG 3' 3'      AGAGTTT   TAAATGC 5'
••• S«r Gin Ly» Ph« Thr •••
••• Ser Gin
Phe Thr ••■
• Silent mutation Base pair substitution results in the same amino acid.
TCTCAAAAHTTTACG	3'	5'	TCTCAAAA	TTTACG	3
AGAGT T TTilAA ATGC	5'	3-	AGAGTT T TE	AAATGC	5'
• Ser Gin Ly» Phe Thr •••			••• Ser Gin Lys	Phe Thr •••	
30 Department of Experimental Biology
MUNI
SCI
Substitution and point mutations
• Frameshift mutations
• Deletions or insertions (not divided by 3) result in translation of incorrect amino acids, stops codons (shorter polypeptides), or read-through of stop codons (longer polypeptides).
5' TCTCAAAAATTTACG 3' 3'      AGAGTTTTTAAATGC 5'
••• Ser Gin Lyt Phe Thr •••
5' 3'
TCTCAA AGAG T T
AAATTTACG 3' TTTAAATGC 5'
Ser Gin
Glu [lie ITyr
31   Department of Experimental Biology
MUNI
SCI
Additional type of Mutations
• null mutation - complete loss of function o often deletion of the gene or part of it.
• tight mutation - clear phenotypic expression
o e.g. complete loss of ability to grow under certain conditions or preventing the formation of a product of a given biochemical pathway.
• leaky mutation - partial activity of the gene product preserved
o e.g. residual activity of the enzyme will allow cells to survive.
• frameshift mutation - changes the reading frame, o usually deletions or insertions.
• polar mutation - affects the expression of neighboring genes, o In operons.
32  Department of Experimental Biology
MUNI
SCI
Mutation and structural abberations
B   E   D   C F
A   B   C   D   D   E F
Breakage of a chromosome can produce variety of arrangements.
inversion Duplication A   B   C   D   E F
Deletion A   B   D E
Insertion CD   L   E F
Translocation ABCDEF ABCopq
I   m   n   o   p q
I   m   n   D   E F
• Chromosomal structural changes usually occur during meiosis , formation of egg or sperm cells, in early fetal development, or in any cell after birth.
• Pieces of DNA can be rearranged within one chromosome or transferred between two or more chromosomes.
• Some changes cause health problems, while others may have no effect on a person's health.
Department of Experimental Biology
https://biologyboom.com/change-in-chromosome-structure/
https://medlineplus.gov/genetics/understanding/mutationsanddisorders/structuralchanges/
MUNI
SCI
Mutation and structural abberations
Inversion
Paracentric Inversion
An inversion occurs when a chromosome breaks in two places; the resulting piece of DNA is reversed and re-inserted into the chromosome.
Genetic material may or may not be lost as a result of the chromosome breaks.
An inversion that includes
o the chromosome's constriction point (centromere) is
called a pericentric inversion, o the long (q) arm or short (p) arm and does not
involve the centromere is called a paracentric
inversion.
34 Define footer - presentation title / department https://medlineplus.gov/genetics/understanding/mutationsanddisorders/structuralchanges/
.Reinserted J Piece of DNA with Centromere
MUNI
SCI
Mutation and structural abberations
Duplications
Duplication
Duplications occur when part of a chromosome is abnormally copied (duplicated). This type of chromosomal change results in extra copies of genetic material from the duplicated segment.
Chromosome
>Duplicated Genetic Material
itional Library of Medicine
35 Department of Experimental Biology
https://medlineplus.gov/genetics/understanding/mutationsanddisorders/structuralchanges/
MUNI
SCI
Mutation and structural abberations
• Deletions
Deletions occur when a chromosome breaks and some genetic material is lost. Deletions can be large or small, and can occur anywhere along a chromosome.
Deletion
Breaks in Chromosome
36 Department of Experimental Biology
https://medlineplus.gov/genetics/understanding/mutationsanddisorders/structuralchanges/
MUNI
SCI
Mutation and structural abberations
Translocations
• A translocation occurs when a piece of one chromosome breaks off and attaches to another chromosome. This type of rearrangement is described as balanced if no genetic material is gained or lost in the cell.
37 Department of Experimental Biology
Chromosome B
Chromosome A
U.S. National Library of Mediane
https://medlineplus.gov/genetics/understanding/mutationsanddisorders/structuralchanges/
MUNI
SCI
Mutation and structural abberations
• Translocations
• A translocation occurs when a piece of one chromosome breaks off and attaches to another chromosome.
• If there is a gain or loss of genetic material, the translocation is described as unbalanced.
38 Department of Experimental Biology
Unbalanced Translocation
https://medlineplus.gov/genetics/understanding/mutationsanddisorders/structuralchanges/
MUNI
SCI
Are mutatins good or bad?
It depends.
Mostly harmful with a negative effect on the function of the gene product.
The damaged gene product may not always be a protein, but also RNA (tRNA, rRNA, atd.).
Mutations can also damage non-coding but important signal sequences.
Most mutations do not have a significant effect on the survival of the organism - they are neutral.
Rarely, the mutation has a positive effect on the survival and reproduction of the organism.
The accumulation of these beneficial mutations will allow the organism to develop into a changing with the environment.
MUNI
Department of Experimental Biology https://open.lib.umn.edu/evolutionbiology/chapter/5-8-using-the-genetic-code-2/
O 0 J.
Impact of mutation depeneds on many aspects
• Mutations that alter the meaning of the codon ("missense mutations") are the most common.
• Lead to the substitution of one amino acid for another in a protein molecule.
• If the mutation replaces the original amino acid with a chemically related amino acid -usually without serious consequences (conservative substitution).
• The consequences are serious if they change the way the protein is composed or the structure active site (non-conservative substitution).
Conservative substitution
Radical replacement
Original protein
40 Department of Experimental Biology
dna mutation g ca -» gga
Amino acid
change ala -» gly
DNA mutation G CA -» GAA
Amino acid
change ALA -»GLU
Mutated
PROTEIN
Original
PROTEIN
Mutated
PROTEIN (glutanine has extra
negative charge and folds incorrectly)
MUNI
SCI
Classification of Mutations
• According to the method of formation:
Spontaneous - occur in the absence of known mutagen, without apparent external cause.
• Consequence of metabolic disorders in the body.
• Consequence of DNA replication errors.
• Consequence of the presence of an unknown mutagenic substance in the environment.
Induced - occur in the presence of known mutagen Physical. ^
• Chemical. I MUTAGENS
• Biological factors. J
MUNI
41   Department of Experimental Biology r> r» t
• Spontaneous mutations
MUNI
42  Department of Experimental Biology _ _ T
O 0 J.
Base pairing- tautomerization
(.i) Stnntf.ird base pairing arrangements
ImM—H-\ / N—C
*  V J
O H
Thymine (keto) Adenine (amino)
0---u—n
\
Cylosirie (amino) (b) Anomalous base-pairing arrangements
>-<
H—C        jííl-M — Hi
H'—C J
Guanine (ketoj
—H—W
Standard base-pairing arrangements of the canonical nucleotide isomers.
Anomalous base-pairing arrangements of the tautomers.
Natural cause of certain genome instability.
Base transitions to different forms change their pairing specificity.
When tautomerization occurs during replication, the DNA sequence will be "misread", and anomalous base-pairing will occur: such as C* with A, or T*.
43 Department of Experimental Biology
https://www.researchgate.net/publication/334974633_Unified_Physics_and_the_Entang lement Nexus of Awareness
MUNI
SCI
Mutation caused by mismatch wobbl base pairing
a) b) c) d>
Guanine Mismatched GT GC-to-AT transition
wobble pairs      base pair after mutation produced
with T replication after next DNA
replication
5'_3' DNA
ACGTC replication
TGCAG
3'-5'
Parentat DNA
44 Department of Experimental Biology
AC T G
—i—i—i—i—r~ ACGTC TGTAG
DNA
replication
ACGTC TGCAG
First-generation progeny
ACGTC
TGCAG W.ldtype
AC ATC TGTAG
ACGTC TGCAG
Mutant
Wild type
ACGTC
TGCAG  Wild type
i__
Seco nd -generati o n progeny
MUNI
SCI
Insertion and deletion by DNA looping-out
New strand
5
5
3'
5'
3J
i i i i i i i II AGTCGCATAGTT TCAGCGTATCAAAAACGTCGATC Template strand 3f----' I I I I I I M I - ' ' I I M I '----5'
I I I I I I I I I I I I AGTCGCATAGTT
TCAGCGTATCAA AACGTCGATC
I I I I I I.....IAS Ml
- -^ty--
Looping out of
template strand
5'
One base deletion on new strand
3'
i i I I I I I I I I i i i,i I I I I I I I I '
AGTCGCATAGTTTTGCAGCTAG TCAGCGTATCAA AACGTCGATC
...........B......... -r
3'
5'
3'
Looping out of new strand 3
AGTCGCATAGTTTT TCAGCGTATCAA AAACGTCGATC
5'
One base insertion on new strand
ft..........-3'
i i i i I
AGTCGCATAGTT TTTGCAGCTAG TCAGCGTATCAAAAACGTCGATC
5'
45 Department of Experimental Biology
MUNI
SCI
Hot spots in repetitions of DNA
120
M E	100
~	
E	BO
5	
E	60
0	
	
QJ a	40
E	
D Z	20
	0
B	
	2000
0)	1800
B 0	1600
1	1400
	
£	1200
B	1000
i_ fib	800
iqui	600
Z	400
	200
Germiine mutations
MED
u.iUíjL
A. \ L
1J
... J JjJ.
(Í5ÍL
I
lill..,J,
CVJ M
Amino acids
Somatic mutations
firTT?>
.j!kULlli
Amino acids
TA
• The distribution of mutations in the gene or genome is not uniform.
• Certain DNA sequences have a higher risk of being affected by mutations „hot spots".
I • Hot-spots - DNA sequences with increased probability of mutation events.
• The replication apparatus has some problems with repetitive sequences.
• Areas of tandem repeats are unstable, often occurence of insertions and deletions.
pr
DBD
Tet
Reg
46 Department of Experimental Biology
Potential clinical relevance (p53).
MUNI
SCI
Replication polymerase slippage
Due to assembly of loops, part of the template can be copied during replication repeatedly or can be omitted replication slippageTpolymerase slippage".
Trinucleotide expansion: neurological diseases (Huntigton's disease, expansion of trinucleotide CAG).
5' 3'
5' 3'
3'
5' 3'-
Expansion
3'
-■-►3'
Replication
5'
Strand dissociation
5'
^^^Contraction
5 I—i 5' 3'H
3' 5'
47 Department of Experimental Biology
MUNI
SCI
Deamination
• Depurination
• Common; A or G are removed and replaced with a random base.
• Deamination
• Amino group is removed from a base (C —► U); if not replaced U pairs with A in next round of replication (CG —► TA).
• Prokaryote DNA contains small amounts of 5mC; deamination of 5mC produces T (CG TA).
• Regions with high levels of 5mC are mutation hot spots.
48 Define footer - presentation title / department
MUNI
SCI
Deamination
b)
NH,
N^4 3 5
N
CH,
H
Methyl group
Deamination
5-methylcytosine (5mC)
H
N3 4 5
2 1.5 N
CH,
H
Thymine (T)
49 Department of Experimental Biology
MUNI
SCI
Induced mutations
rimental Biology
Mutagens
Mutagen - any agent, physical or environmental, that can induce a genetic mutation or can increase the rate of mutation.
Mutagens can be physical mutagens, chemical mutagens, or biological mutagens.
The ability of a substance to induce the alterations in the base pairs of DNA or mutation is known as mutagenicity.
Physical
Chemical
Heat & Radiation
Base analogs
I nte rca I at i ng age nts
Metal ions
Alkylatingagents
	Viruses
Biological !	5 Bacteria
	1 Transposons&
Insertion sequ
51   Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
MUNI
SCI
Mutagens
Physical Mutagens,
Biological Mutagens
Ionizing radiation X-Rays, y-Rays
■Ô*
^ y-Rays
Non-ionizing radiation UVRays, Heat
\r\r\r
■
Virus
Alkylating agents
Base Analogues ,
> Acridinedyes
■
De-aminating Agents
Metal Ions
(EMS, MMS, nitro gen mustard etc)
(5BU, 2-AP, TMU etc)
(Acraflavin, acridine orange, ethidium bromide etc.).
(some bisulfite etc)
(Ni, Cr, Co, Cd, As, etc)
Bacteria
Transposonsand T-DNA
■
Targeted Genome editing
r^TALENS ^CRISPR >ZFN
■ o
52  Department of Experimental Biology
Journal of Applied Biology & Biotechnology Vol. 9(04), pp. 162-169, July, 2021
MUNI
SCI
Physical mutagens
• Ionizing radiation - X-ray, gamma, cosmic, causes DNA breaks.
• Non-ionizing radiation (UV) - specific absorption at wavelength 260 - 280 nm, formation of thymine dimers.
• Heat - the phosphodiester bonds break in DNA when heated above 95°C resulting in breakage of DNA strand.
Electromagnetic Radiation Spectrum
io»
Wavelength (Metres) -I-1-h-
10«      10«      10*      10*      10»      lO*     10*      10*      10*    10«     lO"    10"   10* 10"
H-       I        I —I-1-       I       I —t-1-1-1-1-h-
Quentum (Photon) Energy (eV) 10*      10»     10*    10'« 10"
-I-1-1-1-r—
I
t
R».dioWov»s
H-1-
. UKrowam ;.]
Human Boöy He« I Win»*«
Radar -1-
XRays
103       10* 104
Frequency (H2)
10*
10* 10!
7 í xtC V
Gamma Rays
CowncRayj • —-
-h-
10«     10»     10»   10» 10»
35x10-"M
Non-Ionising Radiation
>
53 Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
IOP Conf. Series: Materials Science and Engineering 374 (2018) 012009
MUNI
SCI
Ionizing radiation - physical mutagens
• Ionizing radiation - X-ray, Gamma and Cosmic radiations.
• Shorter wavelengths and greater energy than visible light, which penetrates deep into tissues, crashes into atoms, releases electrons, positively charged radicals and ions are formed, which provoke the formation of other ions (ionization process).
• These radiations exert a lethal (i.e., killing the cell) or sub-lethal (i.e., changing the functioning of the cell) effect by directly damaging the DNAorthe nucleotides by: o inducing-cross-linking of DNAor protein,
o breaking of chromosomes,
o breaking of strands or chromosomal loss,
o molecularly deletion of bases/DNA strand breakages.
54 Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
MUNI
SCI
UV radiation - physical mutagens
• Lower energy than ionizing radiation.
• Does not cause ionization.
• Penetrates only into the upper layers.
• Strong mutagen in unicellular organisms.
• The energy of radiation is captured by atoms whose electrons pass to the excited state - increase in the reactivity of atoms and molecules, leading to mutations in DNA.
Maximum mutagenic effects at 254 nm (maximum absorption).
55 Department of Experimental Biology
https://genesiswatertech.com/blog-post/types-of-ultraviolet-uv-lamps/
MUNI
SCI
UV radiation - physical mutagens
• UV radiations are of three types - UV A, UV B, and UV C.
• UV-A is the UV rays with a wavelength of 320nm (near-visible range) and is known to result in dimerization of pyrimidines. This kind of pyrimidine dimerization results in alteration of the DNA structure which averts the formation of the replication fork during the process of replication. Such dimerization may lead to health issues.
• UV-B has a wavelength of 290-320nm and highly lethal to DNA.
• UV-C radiations have a wavelength of 180-290nm and are the most lethal as well as carcinogenic. UV-C radiations are majorly absorbed by the ozone layer.
56 Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
MUNI
SCI
UV radiation - physical mutagens
After absorption of UV, Pyrimidines become ^2 very reactive and change themself to A   H 1^ /
pyrimidine hydrates and then to the dimers. a+H0 N"
M 1 >
H
Pyrimidine dimers results in formation of the        I I replication fork and block of replication. „ x . „ ± .   u ±
Cytosine Cytosine hydrate
H     ^-c        CH3 H3C,
I      II +
S!l^     H H"
Thymine
?
Thymine
UV
CH3 CH3
H.
H
O N
H
r
H
I
Thymine dimer
57 Define footer - presentation title / department
Thymine dimers induced by UV light.
J U N I
SCI
Chemical mutagens
• Chemical mutagens influence the structure of DNA: o Oxidizing agents (peroxides, oxygen radicals).
o Deaminating substances (nitrites), o Alkylating agents (mustard gas), o Intercalation agents (acridines).
o Aromatic amines (benzidine, naphthylamine) damaging the cellular
apparatus for even distribution genetic information in cell division, o Colchicine
• Chemical mutagens can cause mutations:
o Regardless of whether DNA replication takes place (e.g. alkylating agents, nitric acid), o Only during DNA replication (base analogues, acridine dyes).
58 Department of Experimental Biology
MUNI
SCI
Base analogues - chemical mutagens
• 5-bromuracil is a thymine analogue - structurally similar to the normal bases.
• Capable of non-standard pairing (tautomerism).
• Incorporated into DNA during replication.
• 5-bromuracil induces transitions in both directions (different charge distribution increases frequency of tautomeric rearrangement - other pairing).
0 |_j
0
H
H
R
O
R
O---H-N
5-bromuracil
H
(5-BU)
5-BrU (keto)
Adenine
5-BrU (enol)
Guanine
59 Department of Experimental Biology
https://pt.wikipedia.org/wiki/5-Bromouracil
MUNI
SCI
Base analogues - chemical mutagens
Due to structural similarities these agents with the DNA bases, base analogs get incorporated in the DNA structure during the process of replication.
Aminopurine is similar to adenine and can form a base pair with C orT (though base pairing with C is rare).
5-Brornouracil
HC
\ / N-C
2-Arninopurine
h
N C hX n
ch I
-n h
a) Base-pairing of 5-bromouracil in its normal state
H
Br
\
,c—c
, c / \\ Attachment XL
of base to sugar
5-bromouracil (behaves like thymine; normal state)
/
H
^ of base to sugar
O
Attachment
Adenine (normal state)
b) Base-pairing of 5-bromouracil in its rare state
H-c
Br
\
t
\
0-H..-0
N-
w
0-
■H-N
\
5-bromouracil (behaves like cytosine; rare state)
Guanine (normal state)
60 Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
MUNI
SCI
Base analogues - chemical mutagens
• Keto form of the 5-bromouracil replaces thymine, whereas enol tautomeric forms a replaces guanine.
Upon replication DNA containing 5-Bromouracil changes base pair from A-T to G-C or from a G-C to an A-T.
c) Mutagenic action of 58U
AT-to-GC transition mutation AdrJSBU
DNA
replication
GC-to-AT transition mutation
^H&I^H       Add SBU
_G_
DNA
replication
61   Department of Experimental Biology
T A
5BU
A
SBU shifts to rare stares
SBU incorporated in normal slate
C G
DNA
replication'
■■■■	
	
	1
5BU returns to normal state
SBU incorporated in rare state
DNA
replication
SBU
A
5BU shifts back
to normal $tate
DNA
re plica lion*
C G
Transition mutation (instead of ATT il is CG)
DNA
replication x
T A
Transition mutation [instead of GC, it is AT)
MUNI
SCI
Base modifying agents - chemical mutagens
Base modifying agents affects DNAthat is not replicated:
• Intercalating agents
• Nitric acid
• Hydroxylamine
• Sodium bisulfate
• Alkylating agents
62  Department of Experimental Biology
MUNI
SCI
DNA intercalating agents - chemical mutagens
• DNA Intercalating agents are the molecules that have a hydrophobic heterocyclic ring structure and resemble the ring structure of base pairs.
• These agents place themselves in the DNA helix, which eventually interferes with the replication, translation, and transcription resulting in mutation, most commonly frameshift mutation.
• Ethidium bromide, proflavine, acridine orange, actinomycin D, or daunorubicin, etc. are some of the common intercalating agents.
• Daunorubicin along with Epirubicin and Mitoxantrone are some of the common anti-cancer or antineoplastic drugs.
63 Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
MUNI
SCI
DNA intercalating agents - chemical mutagens
Ettiidiurn
0 OH O
Adripmycin {R = OH)
64 Department of Experimental Biology
https://www.hoelzel-biotech.com/en/infothek/nucleic-acid-detection/ |y|     |j j
https://www.sciencedirect.com/topics/medicine-and-dentistry/intercalating-agent
SCI
Deaminating
agents - chemical mutagens
• Deaminatnig agent causes oxidative deamination of adenine, guanine and cytosine. Amino groups are thus converted into keto groups.
• Guanine —► xanthine, which pairs with cytosine, thus guanine deamination is not mutagenic.
• Cytosine —► uracil, which is paired with cytosine.
• Adenine —► hypoxanthine, which is paired with cytosine.
65 Department of Experimental Biology
Original base
a)
1> H^Nv
t
OR'
N---U \-H
/
n c
2)
Guanine^ H
h Vh
o
dR 0 Cytosine
n h
3)
H
\
HV^N\ N-H N
Adenine
Mutagen
Nitrous acid (HN02) -►
Nitrous acid (HNO,) -►
Nitrous acid (HN02) -►
Modified base    Pairing partner
H
/
p —h   n h
•c c —c
--{' n-h—n NC-h
dR        \        / \ /
/
N — C
h o Xanthine
\ / C —n
O* dR Cytosine
h        0-—h-n
NdR
dR O Uracil
Adenine
P — h-n h
C c —C
u // \ // \
n   h—n C-h
n=< C—n
H r/ dR
Hypoxanthine Cytosine
Predicted transition
None
CG—►TA
AT—>GC
MUNI
SCI
Deaminating agents - chemical mutagens
• Bisulfide has the same effect as nitric acid, deamination of cytosine to uracil, GC —► AT.
• 5mC can not be deaminated.
5'
Genomic DNA
■c
G
C G
m
G-G
treatment with bisulfite
u
I
u — c
■u
HSQ," Sulfon&tion
OH
3 deamlfiation
HSQ,~
' 0H-
0 _ Alküli 3 desulfonation
Cytosine sulfonate
Uracil sulfonate
B
Uracil
	Original sequence   Sequence after bisulfite treatment
Unmethylated DNA Methylated DNA	A-T-C-G-G-T-C-A-T-C-G-C-A-T              A-T-U -G-G-T-U - A-T-U-G-U -AT A-T-C-G-G-T-C-A-T-C-G-C- A-T             A-T-C-G -G-T-U-A-T -C-G -U-A-T
PCR (1st cycle) I
m
5' -u — u — c — u
---A - -A- - G-**~A
I
PCR (amplification)
I
■T+--T----C
A "-- A----G-
t-
A
sequencing
+ bisulfite C
C T
		1— Ť O		
		c		
		g		
		- £-		
				
		conv		
66 Department of Experimental Biology
June 2005Biochemistry (Moscow) 70(5):596-603 Clinical Chemistry, 2009, 55(8), 1471-83.
Hydroxylating agents - chemical mutagens
• Hydroxylation of an amino group of cytosine.
• Hydroxyl-amino-cytosine forms pair with adenine.
• Transition CG —► TA.
Original base h Vh dR 0 Cytosine	Mutagen Hydroxylamine (NHjOH)	Modified base   Pairing partner h-os » H          N-—H-N Z*1"^" dR         o H Hydroxylaminocytosinc Adenine	Predicted transition CG—*-TA
			
67 Department of Experimental Biology
MUNI
SCI
Alkylating agents - chemical mutagens
Alkylating agents induce alkyl groups (methyl, ethyl) in DNA resulting in altered pairing abilities or create inter-strands cross-links.
The introduction of the alkyl groups increases ionization that results in base-pairing errors and eventually inducing gaps in the DNA strand.
Some of the common alkylating agents are ethylnitrosourea, mustard gas, vinyl chloride, Methylhydrazine, Busulfan, Carmustine, lomustine, Dimethyl sulfate, Temozolomide, Dacarbazine, Ethyl ethane sulfate, and Thio-TEPA.
Induces all types of mutations (transitions, transversions, frameshift mutations and chromosomal aberrations).
CH3-CH2—0-S02—CH3 CI-CH2-CH2-S-CH2-CH2-CI
Ethylmethansulfonate Di-(2-chlorylethyl) sulfide
MuStard 935 MUNI
68 Department of Experimental Biology _ _ T
O 0 J.
Alkylating agents - chemical mutagens
• By alkylation of guanine mustard gas causes changes that block replication and cell division (consideration of use in the treatment of cancer).
• Though during the DNA repairing process, alkylated bases can be removed from the DNA by the depurination process. Depurination is a non-mutagenic process.
Original base	Mutagen	Modified base   Pairing partner	Predicted transition
c)			
N=c\ N-H /	Methylmethane sulfonate (MMS) (alkylating agent) -*■	Hy^-\     /0_CM' ^ ,CH3 I >-< /c"\ „R'^^-\        iN—H-N C-H N-H.....0 dR / H 06-Methylguanine Thymine	GC—*AT
H Guanine			
69 Department of Experimental Biology https://www.biologyonline.com/dictionary/mutagen
Biological mutagens
• Viruses - can insert its viral DNA into the genome.
• Certain viruses, e.g. Rous sarcoma virus, hepatitis B virus, HIV, Epstein-Barr virus, directly influence the genetic material in cells, changing the functioning of genes and triggering cancers. Thus, it can be said, that viruses can be mutagenic.
• Bacteria - certain inflammation-inducing bacteria like Helicobacter pylori produces reactive oxygen species that results in DNA damage and reduced DNA repair. This raises the likelihood of the mutation.
70 Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
MUNI
SCI
Biological mutagens
• Transposons and Insertion sequences (IS) - are units of DNA that undertake self-directed relocation/multiplication of the DNA fragment.
• Both IS and transposon are also known as jumping genes as they move from throughout the DNA.
• The addition of transposons into chromosomal DNA interrupts the functionality of the genes.
• Three types of transposons are usually found:
o Replicative transposons - transposons that retain the original locus and translocate its copy, o Conservative transposons - the original transposon translocates itself, o Retrotransposons transpose - translocate via RNA intermediates.
71   Department of Experimental Biology
https://www.biologyonline.com/dictionary/mutagen
MUNI
SCI
Ames test
American bacteriologist.
In the late 60s, he is studying mutations in Salmonella at Berkeley .
He observed that mutations in certain genes prevent the growth of bacteria on a Petri dish.
In this strain there may be another mutation that allows the ability to restore the grow - a bacterial colony is formed on the dish.
Bruce Ames
More colonies, the greater the mutagenic capacity of the substance used, It can be quantified.
The reversal rate reflects the strength of the mutagen.
72  Department of Experimental Biology
https://gairdner.org/award_winners/bruce-n-ames/
MUNI
SCI
Ames test
Ames Test is an inexpensive method used to screen possible carcinogens and mutagens.
Histidine auxotroph Salmonella typhimurium (requires Histidine to grow) are mixed with rat liver enzymes and plated on media lacking histidine.
Liver enzymes are required to detect mutagens that are converted to carcinogenic forms by the liver (e.g., procarcinogens).
Test chemical is then added to medium.
Control plates show only a small of revertants (bacteria cells growing without Histidine).
Plates inoculated with mutagens or procarcinogens show a larger of revertants.
Auxotroph will not grow without Histidine unless a mutation has occurred.
MUNI
73 Department of Experimental Biology https://slidetodoc.com/chapter-7-a-dna-mutation-and-repair-mutation/
O 0 J.
Arnes test
Negative result
74 Department of Experimental Biology
https://slidetodoc.com/chapter-7-a-dna-mutation-and-repair-mutation/
MUNI
SCI
THANK YOU FOR YOUR ATTENTION
The Paramecium Pallor _
75 Department of Experimental Biology
Sharing mutation stories was a DNA camping tradition.
https://cz.pinterest.com/pin/515451119831203910/
MUNI
SCI