This document discusses genetic mutations and DNA repair. It defines mutations as heritable changes in genetic material that can provide genetic variation and be the basis for evolution. Mutations can be caused spontaneously during DNA replication or cell division, or can be induced by environmental mutagens. The majority of mutations are neutral or harmful, with a small percentage being beneficial. Different types of mutations are described, including point mutations, insertions, deletions, and trinucleotide repeats. The effects of mutations on genes and proteins are explained. The timing of mutations as either germline or somatic is an important factor. Causes of spontaneous mutations like depurination and deamination are outlined.

1. GENE
MUTATION
AND DNA REPAIR
By
M.Vharshini
Sri Ramachandra University
1

2. GENETIC MATERIAL
• DNA
– Primary function permanent storage of
information
– Does not normally change
– Mutations do occur
2

3. MUTATIONS
• Mutation
– Heritable change in the
genetic material
– Permanent structural
change of DNA
• Alteration can be passed on
to daughter cells
• Mutations in reproductive
cells can be passed to
offspring
3

4. MUTATIONS
• Mutations
– Provide allelic variation
• Ultimate source of genetic variation
• Foundation for evolutionary change
– Various phenotypic effects
• Neutral
• Harmful
• Beneficial
4

5. MUTATIONS
• Mutations
– Most mutations are neutral
– More likely to be harmful than beneficial
to the individual
• More likely to disrupt function than improve
function
5

6. MUTATIONS
• Mutations
– Many inherited diseases result from
mutated genes
– Diseases such as various cancers can be
caused by environmental agents known
to
cause DNA mutations
• “Mutagens”
6

7. MODEL ORGANISMS
• Much of our understanding of
mutations is a result of the study of
model organisms
– e.g., Bacteria, yeast, Drosophila, etc.
• Amenable to analysis
• Short generation time, numerous offspring,
etc.
– Often exposed to mutagenic
environmental agents
• Effects of mutations are studied 7

8. TYPES OF MUTATIONS
• Types of mutations
– Chromosome mutations
• Changes in chromosome structure
– Genome mutations
• Changes in chromosome number
– Single-gene mutations
• Relatively small changes in DNA
structure
• Occur within a particular gene
• Focus of study in this chapter
8

9. TYPES OF MUTATIONS
• Mutations involve the permanent
alteration of a DNA sequence
– Alteration of base sequence
– Removal or addition of one or more
nucleotides
9

10. MUTATIONS
• Point mutations
– Change in a single base pair within the
DNA
– Two main types of point mutations
• Base substitutions
– Transition
– Transversion
• Small deletions or insertions
10

11. MUTATIONS
• Two types of base substitutions
– Transition
• Pyrimidine changed to another pyrimidine
– e.g., C  T
• Purine changed to another purine
– e.g., A  G
– Transversion
• Purines and pyrimidines are
interchanged
– e.g., A  C
• More rare than transitions 11

12. EFFECTS OF MUTATIONS
• Mutations within the coding sequence
of a gene can have various effects on
the encoded polypeptide’s amino acid
sequence
– Silent mutations
– Missense mutations
• Included neutral mutations
– Nonsense mutations
– Frameshift mutations
12

13. EFFECTS OF MUTATIONS
• Silent mutations
– Amino acid sequence is not altered
• e.g., CCC  CCG (pro  pro)
– Genetic code is degenerate
– Alterations of the third base of a codon often do not alter
the encoded amino acid
– Phenotype is not affected
13

14. EFFECTS OF MUTATIONS
• Missense mutations
– Amino acid sequence is altered
• e.g., GAA GTA (glu  val)
– Phenotype may be affected
14

15. EFFECTS OF MUTATIONS
• Neutral mutations
– Type of missense mutation
– Amino acid sequence is altered
• e.g., CTT ATT (leu  ile)
• e.g., GAA GAC (glu  asp)
– No detectable effect on protein function
• Missense mutations substituting an amino acid with
a similar chemistry to the original is likely to be
neutral
15

16. EFFECTS OF MUTATIONS
• Nonsense mutations
– Normal codon is changed into a stop
codon
• e.g., AAA  AAG (lys  stop)
– Translation is prematurely terminated
• Truncated polypeptide is formed
– Protein function is generally affected
16

17. EFFECTS OF MUTATIONS
17

18. EFFECTS OF MUTATIONS
18

19. EFFECTS OF MUTATIONS
• Mutations occasionally produce a
polypeptide with an enhanced ability to
function
– Relatively rare
– May result in an organism with a greater
likelihood to survive and reproduce
– Natural selection may increase the
frequency of this mutation in the
population
19

20. MUTATION TYPES
• Genetic terms to describe mutations
– Wild-type
• Relatively common genotype
• Generally the most common allele
– Variant
• Mutant allele altering an organism’s
phenotype
– Forward mutation
• Changes wild-type allele into something else
– Reverse mutation
• “Reversion”
• Restores wild-type allele
20

21. MUTATION TYPES
• Genetic terms to describe mutations
– Deleterious mutation
• Decreases an organism’s chance of
survival
– Lethal mutation
• Results in the death of an organism
• Extreme example of a deleterious
mutation
– Conditional mutants
• Affect the phenotype only under a
defined set of conditions
• e.g., Temperature-sensitive (ts) mutants
21

22. MUTATION TYPES
• Genetic terms to describe mutations
– Suppressor mutation
• Second mutation that restores the wild-type
phenotype
• Intragenic suppressor
– Secondary mutation in the same gene as
the first mutation
– Differs from a reversion
» Second mutation is at a different site
than the first
• Intergenic suppressor
– Secondary mutation in a different gene than the first
mutation
22

23. MUTATION TYPES
• Two general types of intergenic
suppressors
– Those involving an ability to defy the
genetic code
– Those involving a mutant structural gene
23

24. MUTATION TYPES
• Intergenic suppressor mutations involving
an ability to defy the genetic code
– e.g., tRNA mutations
• Altered anticodon region
• e.g., Recognize a stop codon
– May suppress a nonsense mutation in a gene.
– May also suppress stop codons in normal genes.
24

25. MUTATION TYPES
• Intergenic suppressors involving a mutant structural gene
– Usually involve altered expression of one gene that
compensates for a loss-of-function mutation affecting
another gene
• Second gene may take over the functional role of the
first
• May involve proteins participating in a common
cellular function
– Sometimes involve mutations in genetic regulatory
proteins
• e.g., Transcription factors activating other genes that
can compensate for the mutation in the first gene
25

26. MUTATION TYPES
• Mutations occurring outside of coding sequences
can influence gene expression
– Mutations may alter the core promoter sequence
• Up promoter mutations
– Mutant promoter becomes more like the
consensus sequence
– Rate of transcription may be increased
• Down promoter mutations
– Mutant promoter becomes less like the
consensus sequence
– Affinity for regulatory factors is decreased
– Rate of transcription may be decreased
26

27. MUTATION TYPES
• Mutations occurring outside of coding
sequences can influence gene
expression
– Mutations may alter other regulatory
sequences
• lacOC
mutations prevent binding of
the lac repressor
– Lac operon is constituently expressed,
even in the absence of lactose
» Such expression is wasteful
» Such mutants are at a selective
disadvantage
27

28. MUTATION TYPES
• Mutations occurring outside of coding
sequences can influence gene
expression
– Mutations may alter splice junctions
• Altered order and/or number of exons in the
mRNA
28

29. MUTATION TYPES
• Mutations occurring outside of coding
sequences can influence gene
expression
– Mutations may affect an
untranslated region of mRNA
• 5’- or 3’-UTR
• May affect mRNA stability
• May affect the ability of the
mRNA to be translated
29

30. MUTATION TYPES
30

31. TRINUCLEOTIDE
REPEATS
• DNA trinucleotide repeats
– Three nucleotide sequences repeated in
tandem
• e.g., …CAGCAGCAGCAGCAGCAG…
• Generally transmitted normally from parent to
offspring without mutation
31

32. TRINUCLEOTIDE
REPEATS
• Trinucleotide repeat expansion
(TNRE)
– Number of repeats can readily increase
from one generation to the next
– Cause of several human genetic
diseases
• Length of a repeat has increased above a
certain critical size
• Becomes prone to frequent expansion
32

33. TRINUCLEOTIDE
REPEATS
• TNRE disorders
– Fragile X syndrome (FRAXA)
– FRAXE mental retardation
– Myotonic muscular dystrophy (DM)
– Spinal and bulbar muscular atrophy
(SBMA)
– Huntington disease (HD)
– Spinocerebellar ataxia (SCA1)
33

34. TRINUCLEOTIDE
REPEATS
• TNRE disorders
34

35. TRINUCLEOTIDE
REPEATS
• TNRE disorders
– Expansion may be within a coding
sequence of a gene
• Most expansions are of a CAG repeat
• Encoded proteins possess long tracts of
glutamine
– CAG encodes a glutamine codon
• Presence of glutamine tracts causes
aggregation of the proteins
• Aggregation is correlated with the progression
of the disease 35

36. TRINUCLEOTIDE
REPEATS
• TNRE disorders
– Expansion may be in a noncoding region
of a gene
• Two fragile X syndromes
– Repeat produces CpG islands that become
methylated
– Methylation can lead to chromosome compaction
– Can silence gene transcription
• Myotonic muscular dystrophy
– Expansions may cause abnormal changes in RNA
structure
36

37. TRINUCLEOTIDE
REPEATS
• TNRE disorders
– Severity of the disease tends to worsen in
future generations
• “Anticipation”
– Severity of the disease depends on the
parent from whom it was inherited
• e.g., In Huntingdon disease, TNRE likely to occur
if mutation gene is inherited from the father
• e.g., In myotonic muscular dystrophy, TNRE
likely to occur if mutation gene is inherited from
the mother 37

38. TRINUCLEOTIDE
REPEATS
• TNRE disorders
38

39. TRINUCLEOTIDE
REPEATS
• TNRE disorders
– Cause of TNRE is not well understood
– Trinucleotide repeat may produce
alterations in DNA structure
• e.g., Stem-loop formation
• May lead to errors in DNA replication
– TNRE within certain genes alters gene
expression
• Disease symptoms are produced
39

40. CHROMOSOME STRUCTURE
• Altered chromosome structure can
alter gene expression
– Inversions and translocations commonly
have no obvious phenotypic effects
– Phenotypic effects sometimes occur
• “Position effect”
40

41. CHROMOSOME STRUCTURE
• Altered chromosome structure can alter gene
expression and phenotype
– Breakpoint may occur within a gene
• Expression of the gene is altered
– Breakpoint may occur near a gene
• Expression is altered when moved to a new location
• May be moved next to regulatory elements influencing
the expression of the relocated gene
– i.e., Silencers or enhancers
• May reposition a gene from a euchromatic region to a
highly condensed (heterochromatic) region
– Expression may be turned off 41

42. CHROMOSOME STRUCTURE
• Altered chromosome
structure can alter gene
expression and phenotype
– An eye color gene relocated to
a heterochromatic region can
display altered expression
• Gene is sometimes inactivated
• Variegated phenotype results
42

43. SOMATIC VS. GERM-LINE
• The timing of mutations in multicellular
organisms plays an important role
– Mutations may occur in gametes or a
fertilized egg
– Mutations may occur later in life
• Embryonic or adult stages
• Timing can affect
– The severity of the genetic effect
– The ability to be passed from parent to
offspring 43

44. SOMATIC VS. GERM-LINE
• Animals possess germ-line and
somatic cells
– Germ-line cells
• Cells giving rise to gametes
– Somatic cells
• All cells of the body
excluding the germ-line cells
– e.g., Muscle cells, nerve cells,
etc.
44

45. SOMATIC VS. GERM-LINE
• Germ-line cells
– Germ-line mutations can occur
in gametes
– Germ-line mutations can occur
in a precursor cell that produces
gametes
– All cells in the resulting offspring
will contain the mutation
45

46. SOMATIC VS. GERM-LINE
• Somatic cells
– Somatic mutations in embryonic
cells can result in patches of
tissues containing the mutation
• Size of the patch depends on the
timing of the mutation
• Individual is a genetic mosaic
46

47. CAUSES OF MUTATIONS
• Two causes of mutations
– Spontaneous mutations
• Result from abnormalities in biological
processes
• Underlying cause lies within the cell
– Induced mutations
• Caused by environmental agents
• Cause originates outside of the cell
47

48. CAUSES OF MUTATIONS
• Causes of spontaneous mutations
– Abnormalities in crossing over
– Aberrant segregation of chromosomes during
meiosis
– Mistakes by DNA polymerase during
replication
– Alteration of DNA by chemical products of
normal metabolic processes
– Integration of transposable elements
– Spontaneous changes in nucleotide structure48

49. CAUSES OF MUTATIONS
• Induced mutations are caused by
mutagens
– Chemical substances or physical agents
originating outside of the cell
– Enter the cell and then alter the DNA
structure
49

50. CAUSES OF MUTATIONS
•
50

51. CAUSES OF MUTATIONS
• Spontaneous mutations are random
events
– Not purposeful
– Mutations occur as a matter of chance
• Some individuals possess beneficial
mutations
– Better adapted to their environment
– Increased chance of surviving and reproducing
• Natural selection results in differential
reproductive success
– The frequency of such alleles increases in the
population
51

52. CAUSES OF MUTATIONS
• Joshua and Ester Lederberg (1950s)
– Interested in the relationship between mutation
and the environmental conditions shat select
for mutations
• Scientists were unsure of the relationship
• Two competing hypotheses
– Directed mutation hypothesis
» Some scientists still believed that selective conditions
could promote specific mutations
– Random mutation theory
» Mutations occur at random
» Environmental factors affecting survival select for
those possessing beneficial mutations
52

53. CAUSES OF MUTATIONS
• Mutation rate
– Likelihood that a gene will be altered by a new
mutation
– Expressed as the number of new mutations in
a given gene per generation
• Generally 1/100,000 – 1/billion
– 10-5
– 10-9
53

54. CAUSES OF MUTATIONS
• Mutation rate
– Mutation rate is not a constant number
• Can be increased by environmental
mutagens
– Induced mutations can increase beyond
frequency of spontaneous mutations
• Mutation rates vary extensively
between species
– Even vary between strains of the
same species
54

55. CAUSES OF MUTATIONS
• Mutation rate
– Some genes mutate at a much higher
rate than other genes
• Some genes are longer than others
• Some locations are more susceptible to
mutation
– Even single genes possess mutation
hot spots
» More likely to mutate than other
regions
55

56. CAUSES OF MUTATIONS
• Mutation frequency
– Number of mutant alleles of a given gene divided
by the number of alleles within a population
– Timing of mutations influences mutation
frequency
• Timing does not influence mutation rate
– Mutation frequency depends both on mutation
rate and timing of mutations
– Natural selection and genetic drift can further
increase mutation frequencies 56

57. CAUSES OF MUTATIONS
• Spontaneous mutations: Depurination
– Most common type of naturally occurring
chemical change
– Reaction with water removes a purine (A
or G) from the DNA
• “Apurinic site”
57

58. CAUSES OF MUTATIONS
• Spontaneous mutations: Depurination
– ~10,000 purines lost per 20 hours at
37oC in a typical mammalian cell
• Rate of loss increased by agents causing
certain base modification
– e.g., Attachment of alkyl
(methyl, ethyl, etc.) groups
– Generally recognized by
DNA repair enzymes
• Mutation may result if
repair system fails 58

59. CAUSES OF MUTATIONS
• Spontaneous mutations: Deamination
of cytosines
– Other bases are not readily deaminated
– Removal of an amino group from the
cytosine base
• Uracil is produced
– DNA repair enzymes generally remove
this base
• Uracil is recognized as an inappropriate base
– Mutation may result if repair system fails
• Uracil hydrogen bonds with A, not G
59

60. CAUSES OF MUTATIONS
• Spontaneous mutations: Deamination
of cytosines
– Methylation of cytosine occurs in many
eukaryotic species as well as prokaryotes
– Removal of an amino group from the 5-
methyl cytosine produces thymine
– DNA repair enzymes cannot determine
which is the incorrect base
• Hot spots for mutations are produced
60

61. CAUSES OF MUTATIONS
• Spontaneous mutations: Tautomeric shifts
– Common, stable form of T and G is the keto
form
• Interconvert to an enol form at a low rate
– Common, stable form of A and C is the amino
form
• Interconvert to an imino form at a low rate
61

62. CAUSES OF MUTATIONS
• Spontaneous mutations: Tautomeric
shifts
– Enol and imino forms do not conform to
normal base-pairing rules
• AC and GT base pairs are formed
62

63. CAUSES OF MUTATIONS
• Spontaneous mutations: Tautomeric
shifts
– Tautomeric shifts immediately prior to
DNA replication can cause mutations
• Resulting mismatch could be repaired
• Mutation may result if repair system fails
63

64. CAUSES OF MUTATIONS
• Hermann Muller (1927)
– Showed that X rays can cause induced
mutations
• Reasoned that a mutagenic agent might form
defective alleles
• Experimental approach focused on formation
and detection of X-linked genes in Drosophila
melanogaster
64

65. CAUSES OF MUTATIONS
• The public is concerned about
mutagens for two important reasons
– Mutagenic agents are often involved in
the development of human cancers
– Avoiding mutations that may have
harmful effects on future offspring is
desirable
65

66. CAUSES OF MUTATIONS
• An enormous array of agents can act
as mutagens
– Chemical agents and physical agents
66

67. CAUSES OF MUTATIONS
• Certain non-mutagenic chemicals can
be altered to a mutagenically active
form after ingestion
– Cellular enzymes such as oxidases can
activate some mutagens
• Certain foods contain chemicals acting
as antioxidants
– Antioxidants may be able to counteract
the effects of mutagens and lower cancer
rates 67

68. CAUSES OF MUTATIONS
• Mutagens alter DNA structure in
various ways
– Nitrous acid (HNO3) replaces amino
groups with keto groups
• -NH2  =O
• Can change cytosine
to uracil
– Pairs with A, not G
• Can change adenine
to hypoxanthine
– Pairs with C, not T 68

69. CAUSES OF MUTATIONS
• Mutagens alter DNA structure in
various ways
– Alkylating agents covalently attach methyl
or ethyl groups to bases
• e.g., Nitrogen mustards, ethyl
methanesulfonate (EMS)
– Appropriate base pairing is disrupted
69

70. CAUSES OF MUTATIONS
• Mutagens alter DNA structure in
various ways
– Some mutagens directly interfere with the
DNA replication process
– e.g., Acridine dyes such as proflavin
• Flat, planar structures interchelate into the
double helix
– Sandwich between adjacent base pairs
• Helical structure is distorted
• Single-nucleotide additions and deletions can
result 70

71. CAUSES OF MUTATIONS
• Mutagens alter DNA structure in
various ways
– Some mutagens are base analogs
• e.g., 2-aminopurine
• e.g., 5-bromouracil (5BU)
• Become incorporated into daughter strands
during DNA replication
71

72. CAUSES OF MUTATIONS
• Mutagens alter DNA structure in
various ways
– Some mutagens are base analogs
• 5-bromouracil (5BU) is a thymine
analog
– Incorporated in place of thymine
• 5BU can base-pair with adenine
– Can tautomerize and base-pair with
guanine at a relatively high rate
• AT  A5BU  G5BU  GC
– Transition mutations occur
72

73. CAUSES OF MUTATIONS
• Mutagens alter DNA structure in various
ways
– DNA molecules are sensitive to physical agents
such as radiation
• e.g., Ionizing radiation such as X rays and gamma
rays
– Short wavelength and high energy
– Can penetrate deeply into biological materials
– Creates “free radicals”
» Chemically reactive molecules
– Free radicals alter DNA structure in a variety of ways
» Deletions, single nicks, cross-linking, chromosomal
breaks
73

74. CAUSES OF MUTATIONS
• Mutagens alter DNA structure in various ways
– DNA molecules are sensitive to physical agents
such as radiation
• e.g., Nonionizing radiation such as
UV light
– Contains less energy
– Penetrates only the surface of material
such as the skin
– Causes the formation of thymine dimers
– May be repaired through one of numerous
repair systems
– May cause a mutation when that DNA
strand is replicated 74

75. CAUSES OF MUTATIONS
• Many different kinds of testes can
determine if an agent is mutagenic
– Ames test is commonly used
• Developed by Bruce Ames
– Uses his-
strains of Salmonella
typhimurium
• Mutation is due to a point mutation rendering
an enzyme inactive
– Reversions can restore his+
phenotype
• Ames test monitors rate of reversion
mutations
75

76. CAUSES OF MUTATIONS
• Ames test
– Suspected mutagen is mixed with rat liver
extract and his-
Salmonella typhimurium
• Rat liver extract provides cellular
enzymes that may be required to
activate a mutagen
– Bacteria are plated on minimal
media
• his+
revertants can be detected
• Mutation frequency calculated
– Compared to control 76

77. DNA REPAIR
• Most mutations are deleterious
– DNA repair systems are vital to the survival
– Bacteria possess several different DNA repair
systems
• Absence of a single system greatly
increases mutation rate
– “Mutator strains”
– Humans defective in a single DNA
repair system may manifest various
disease symptoms
• e.g., Higher risk of skin cancer
77

78. DNA REPAIR
• Living cells contain several DNA repair
systems
– Able to fix different types of DNA alterations
78