DNA mutations http://www.ncbi.nlm.nih.gov/books/NBK2 1897/ 1 - - PowerPoint PPT Presentation

DNA mutations

http://www.ncbi.nlm.nih.gov/books/NBK2 1897/

1

Types of mutations

• Micromutation that involve small regions of the DNA
• Macromutations that involve the chromosomes as a

whole

2

DNA micromutations

• Single-base mutations can result in

different types of mutations such as: –missense: a change of an amino acid –nonsense: premature termination of protein synthesis –frameshift: altering the reading of amino acid sequence –silent: does not lead to any change at the protein level, but contributes to genetic variability among individuals

3

4

DNA micromutations (cont.)

• Translocations, that bring different regions of gene

segments together,

• Deletions of a few nucleotides to long stretches of

DNA,

• Insertions and duplications of nucleotides or long

stretches of DNA, and

• Inversion of DNA segments

5

Causes of DNA mutation

• DNA mutations can arise spontaneously or induced
• Spontaneous mutations are naturally occurring

mutations and arise in all cells

• Induced mutations are produced when an organism

is exposed to a mutagenic agent, or mutagen

6

Spontaneous mutations

• Spontaneous mutations arise from a variety of

sources, including errors in DNA replication and spontaneous lesions

7

Errors in DNA replication

• An error in DNA replication can occur when an

inaccurate nucleotide pair (say, A-C instead of G-C) forms in DNA synthesis, leading to a base substitution

• Each of the bases in DNA can appear in one of

several forms, called tautomers

8

9

Effect of tautomers

• Following DNA replication, tautomers lead to either

transition or transversion mutations

10

Transitions

• A purine substitutes for a purine or a pyrimidine for a

pyrimidine

11

Transversions

• In transversion mutations, a pyrimidine substitutes

for a purine or vice versa

• In this case, a replication error would require

mispairing of a purine with a purine or a pyrimidine with a pyrimidine

12

Other errors of DNA replication

• Frameshift mutations
• Deletions and duplication

13

Frameshift mutations

• These mutations often occurred at repeated

sequences

• Such mutations change the reading "frame" of

codons and leads to changes in the amino acid sequence of the produced protein

14

Deletions and duplications

• Large deletions (more than a few base pairs) often
• ccur at repeated sequences
• Duplications of segments of DNA have also been
• bserved
• Like deletions, they often occur at sequence repeats

15

Spontaneous lesions

• Spontaneous lesions are naturally occurring type of

DNA damage that can generate mutations

16

Types of spontaneous lesions

• Depurination
• Deamination
• Oxidatively damaged bases

17

Depurination

• Cleavage of the

glycosidic bond between the base and deoxyribose

18

Deamination

• The deamination of cytosine yields uracil
• Uracil residues will pair with adenine during replication,

resulting in the conversion of a G-C pair into an A-T pair (a GC→AT transition)

Methylated cytosine

• Methylated cytosine can also be methylated resulting in its

conversion to thymine and leading to a transition mutation

Oxidatively damaged bases

• Active oxygen species such as hydrogen peroxide

(H2O2) are normally produced during metabolism

• They can damage DNA, which results in mutation

21

8-oxodG

• A product of oxidative damage is 8-oxo-7-

hydrodeoxyguanosine (8-oxodG, or GO) product frequently mispairs with A, resulting in a high level of G→T transversions

22

INDUCED MUTATIONS

23

Mechanisms of mutagenesis

• Mutagens induce mutations by at least three

different mechanisms:

– replace a base in the DNA – alter a base so that it specifically mispairs with another base – damage a base so that it can no longer pair with any base under normal conditions

24

Incorporation of base analogs

• Some chemical compounds are similar to the normal

bases of DNA that they are incorporated into DNA in place of normal bases; such compounds are called base analogs

25

Mechanism of mutation

• These analogs can produce mutations by causing

incorrect nucleotides to be inserted opposite to them during replication

26

5-bromouracil

• 5-bromouracil (5-BU) is an analog of thymine
• The normal structure of 5-BU pairs with adenine
• When ionized, 5-BU pairs with guanine
• 5-BU causes transition mutations

27

Specific mispairing

• Some mutagens are not incorporated into the DNA but

can cause specific mispairing – For example, the addition of an alkyl group to guanine leads to the formation of O-6-alkylguanine and results in direct mispairing with thymine, and consequently a GC  AT transitions

28

Intercalating agents

• The intercalating agents

such as proflavin and ethidium bromide are planar molecules that can insert themselves (intercalate) between the stacked nitrogen bases imitating base pairs

• The intercalated agent

can cause single- nucleotide-pair insertions

• r deletions

29

Base damage

• A large number of mutagens damage one or more

bases, so no specific base pairing is possible

30

Ionizing radiation

• Ionizing radiation results in the formation of ionized

and excited molecules that can cause damage to cellular components including DNA

• Many different types of reactive oxygen species are

produced that can

– damage bases, – cause breakage of the N-glycosidic bond (AP sites), or – cause strand breaks

31

Mutagens and carcinogens

• Mutagenicity and carcinogenicity are clearly

correlated when most mutagens are also carcinogens (approximately 90 percent)

32

Tests of mutagenicity

• Rapid tests make use of microbes to test for

mutagenicity

• The most widely used test was developed in the

1970s by Bruce Ames, who worked with Salmonella typhimurium

http://www.ncbi.nlm.nih.gov/books/NBK21788/

33

Features of Salmonella typhimurium

• The Ames test uses a mutant strain of S. typhimurium

– cannot grow in the absence of the amino acid histidine because a mutation has occurred in a gene that encodes

• ne of the enzymes necessary for histidine biosynthesis

– They also carry a mutation that inactivates a DNA repair system – They also carry a mutation that eliminates the protective lipopolysaccharide coating of wild-type Salmonella to enable the entry of many different chemicals into the cell

34

Reversion mutation

• In order for these cells to survive in the absence of

histidine, they must have a mutation that corrects the original mutation that prevented the production

• f the missing enzyme
• This type of mutation is known as reversion, because

this second mutation returns the mutant to the wild- type genotype

• This reversion can happen spontaneously or as the

result of a mutagen

35

What is considered a mutagen?

• A compound must increase the number of colonies

grown in the absence of histidine by double compared to cells grown in the absence of the compound

36

Example

Water Motor oil Alcohol Drug X وبأ سبيش5 شورق 1050439200

37

Role of liver enzymes

• In mammals, chemicals are normally detoxified or broken

down by liver enzymes

• In some cases, the action of liver enzymes can create a toxic
• r mutagenic compound from a substance that was originally

safe

• Ames incorporated mammalian liver enzymes in his bacterial

test system

38

Example

Condition Water Motor oil Alcohol Drug X وبأ سبيش5 شورق

• liver enzymes

10 50 43 9 200 +liver enzymes 12 22 50 35 500

39

Significance of Ames test

• Chemicals detected by this test can be regarded as

potential carcinogens

40

DNA repair mechanisms

Resources

• This lecture
• Campbell and Farrell’s Biochemistry, pp. 263-268
• An Introduction to Genetic Analysis. 7th edition.

Griffiths AJF, Miller JH, Suzuki DT, et al., New York: W.

• H. Freeman; 2000.

http://www.ncbi.nlm.nih.gov/books/NBK22004/

42

DNA repair

• Maintaining genetic stability requires not only an accurate

mechanism of DNA replication, but also mechanisms for repairing DNA damage

• These mechanisms are collectively called DNA repair

43

Repair mechanisms

• Prevention of errors before they happen
• Direct reversal of damage
• Excision-repair pathways
• Postreplication repair

44

PREVENTION OF ERRORS BEFORE THEY HAPPEN

45

Reactive oxygen species

• Some enzymatic systems neutralize potentially damaging

compounds before they even react with DNA

• One example of such a system is the detoxification of reactive
• xygen species and oxygen radicals, which are produced

during oxidative damage to DNA

46

Superoxide dismutase

47

Catalase

48

DIRECT REVERSAL OF DAMAGE

49

Cyclobutane pyrimidine

• Some lesions can be repaired by reversal of

DNA damage

• UV light that hits DNA results in the

formation of a covalent interaction between two adjacent pyrimidine bases forming structures known as cyclobutane pyrimidine dimers, most frequently between two thymines

• This product is a mutagenic photodimer

Photolyase

• Cyclobutane pyrimidine can be

repaired by a photolyase that has been found in bacteria and lower eukaryotes but not in humans

51

8-oxodG

• The enzyme product of the

mutT gene prevents the incorporation of 8-

• xydeoxyguanosine (8-
• xodG) into DNA
• 8-oxodG is formed from free

radical attack of DNA and pairs with A rather than C

52

EXCISION-REPAIR PATHWAYS

53

Types

• General excision repair
• Coupling of transcription and repair
• Specific excision pathways

54

General excision repair

• Also termed nucleotide excision repair
• This system includes the breaking of a

phosphodiester bond on either side of the lesion, on the same strand, resulting in the excision of an oligonucleotide

• This excision leaves a gap that is filled by

repair synthesis, and a ligase seals the breaks

55

The mechanism

• In E. coli, the UvrA protein recognizes the damaged DNA, forms a

complex with UvrB and leads the UvrB subunit to the damage site

• The UvrC protein then binds to UvrB
• Each of these subunits makes an incision (a cut)
• The short DNA is unwound and released by a helicase
• DNA polymerase I then fills in the gap, followed by the action of

DNA ligase

56

57

In human…

• In human cells, the process is more complex than its bacterial

counterpart

• However, the basic steps are the same as those in E. coli
• Defect in this mechanism causes a condition known as

Xeroderma pigmentosum

58

Transcription and repair

• In both eukaryotes and prokaryotes, there is a

preferential repair of the transcribed strand of DNA for actively expressed genes

• RNA polymerase pauses when encountering a lesion
• The general transcription factor TFIIH and other

factors carry out the incision, excision, and repair reactions

• Then transcription can continue normally

59

SPECIFIC EXCISION PATHWAYS

60

DNA glycosylase repair pathway

• DNA glycosylases do not cleave phosphodiester bonds, but

instead cleave N-glycosidic (base-sugar) bonds of damaged bases , liberating the altered base and generating an apurinic

• r an apyrimidinic site, both called AP sites
• The resulting AP site is then repaired by an AP endonuclease

repair pathway

61

DNA glycosylases

• Numerous DNA glycosylases exist
• One, uracil-DNA glycosylase, removes uracil

from DNA

• Uracil residues, which result from the

spontaneous deamination of cytosine can lead to a CT transition if unrepaired

62

AP endonuclease repair pathway

• The AP endonucleases introduce chain breaks by

cleaving the phosphodiester bonds at AP sites

• This bond cleavage initiates an excision-repair

process mediated by an exonuclease, DNA polymerase I, and DNA ligase

63

GO system

• mutM removes 8-oxodG, or GO, lesion

from DNA

• If DNA is replicated,

– mutM first removes the GO lesion – Then, mutY removes the mispaired adenine from the opposite strand, leading to restoration of the correct cytosine by repair synthesis (mediated by DNA polymerase I) and allowing subsequent removal of the GO lesion by the mutM product

64

POSTREPLICATION REPAIR

65

Mismatch repair

• Some repair pathways are capable of recognizing errors even

after DNA replication has already occurred

• One such system, termed the mismatch repair system, can

detect mismatches that occur in DNA replication

66

The mechanism

• Recognize mismatched base pairs.
• Determine which base in the mismatch is the

incorrect one.

• Excise the incorrect base and carry out repair

synthesis

67

The mechanism in details

• MutS binds to mispair.
• MutH and MutL are recruited to form a

complex

• MutH cuts the newly synthesized

(unmethylated) strand, and exonuclease degradation goes past the point of the mismatch, leaving a patch

• Single-strand-binding protein (Ssb) protects the

single-stranded region across from the missing patch

• Repair synthesis and ligation fill in the gap

MutS MutL MutH Ssb

68

But….

• In a G-T mismatch, both are normal bases in DNA
• How can the mismatch repair system determine whether G or

T is incorrect?

69

DNA methylation

• DNA is methylated following replication

by the enzyme, adenine methylase

• However, it takes the adenine methylase

several minutes to methylate the newly synthesized DNA

• The mismatch repair system in bacteria

takes advantage of this delay to repair mismatches in the newly synthesized strand

70

In humans

• The mismatch repair system has also been characterized in

humans

• Two of the proteins, hMSH2 and hMLH1, are very similar to

their bacterial counterparts, MutS and MutL, respectively

71

SOS system

• A large number of mutagens such as ultraviolet light damage
• ne or more bases, resulting in a replication block, because

DNA synthesis will not proceed past a base that cannot specify its complementary partner by hydrogen bonding

• In bacterial cells, such replication blocks can be bypassed by

inserting nonspecific bases. The process requires the activation of a special system, the SOS system

72

Proteins of SOS

• Exactly how the SOS bypass system functions is not clear,

although in E. coli it is known to be dependent on at least three genes, recA, umuC, and umuD

• Current models for SOS bypass suggest that the UmuC and

UmuD proteins combine with the polymerase III DNA replication complex

73

The mechanism

• DNA polymerase III pauses at a

type of damage called a TC photodimer

• This region attracts single-strand-

binding protein (Ssb), as well as the RecA protein, which signals the cell to synthesize the UmuC and UmuD proteins forming the SOS system

• The SOS system allows DNA

polymerization to continue past the dimer

74

SOS system and mutations

• However, the SOS system inserts the necessary number of

bases (often incorrect ones) directly across from the lesion and replication continues without a gap

• SOS system often generates mutations

75

Recombinational repair

• The recA gene also takes part in postreplication repair
• Here the DNA replication system pauses at a UV photodimer
• r other blocking lesion and then restarts past the block,

leaving a single-stranded gap

• In recombinational repair, this gap is patched by DNA cut

from the sister molecule

76

MUTATORS

77

What is a mutator?

• Mutant strains with increased spontaneous mutation rates

have been detected

• Such strains are termed mutators
• In many cases, the mutator phenotype is due to a defective

repair system

• In humans, these repair defects often lead to serious diseases

78

Disease related to DNA repair

Condition Sensitivity Cancer susceptibility Mutated gene Ataxia telangiectasia γ irradiation Lymphomas ATM (cell cycle and DNA repair) Bloom syndrome Mild alkylating agents Carcinomas, leukemias, lymphomas ATM (cell cycle and DNA repair) Cockayne syndrome UV light ERCC6 or ERCC8 (DNA repair) Fanconi anemia Cross-linking agents Leukemias Multiple (response to DNA damage) Xeroderma pigmentosum UV light, chemical mutagens Skin carcinomas and melanomas Nucleotide excision repair HNPCC Colon, ovary Mismatch repair

79

Hereditary nonpolyposis colon cancer (HNPCC)

• Several genes responsible a type of colon cancer have been

identified, including MSH2 and MLH1

• The protein products of the MSH2 and MLH1 genes appear to

have critical roles in the recognition and repair of DNA mismatches

• They are homologous to the mutL and mutS genes in bacteria

and yeast

80

DNA sequencing and PCR

Resources

• This lecture
• Campbell and Farrell’s Biochemistry, pp. 377-380,

384-387

82

What is DNA sequencing?

• DNA sequencing is the process of determining the

exact order of the chemical building blocks, that are the A, T, C, and G bases, that make up the genome

83

Importance of knowing the DNA sequence

• Identification of genes and their localization
• Identification of protein structure and function
• Identification of DNA mutations
• Genetic variations among individuals in health and

disease

• Prediction of disease-susceptibility and treatment

efficiency

• Evolutionary conservation among organisms

84

DNA sequencing of organism genome

• Viruses and prokaryotes first
• This was followed by the determination of the

sequence of human mitochondrial DNA

85

Then simple eukaryotes

• The first eukaryotic genome to be completely

sequenced was that of yeast, Saccharomyces cerevisiae, which comprises approximately 12 million base pairs, distributed on 16 chromosomes

• This achievement was followed by the first complete

sequencing of the genome of a multicellular

• rganism, the nematode Caenorhabditis elegans,

which contains nearly 100 million base pairs

86

Last, but not least…

• In humans, it is determining the sequence of the 3

billion bases

• Determination of the base sequence in the human

genome was initiated in 1990 and completed in May 2006 via the Human Genome Project

• Achieving this goal has helped reveal the estimated

20,000-30,000 human genes within our DNA as well as the regions controlling them

87

Method of DNA sequencing

• Based on premature

termination of DNA synthesis by dideoxynucleotides

88

The process…

• DNA synthesis is initiated from a primer that has

been labeled with a radioisotope

• Four separate reactions are run, each including

deoxynucleotides plus one dideoxynucleotide (either A, C, G, or T)

• Incorporation of a dideoxynucleotide stops further

DNA synthesis because no 3 hydroxyl group is available for addition of the next nucleotide

89

Generation of fragments

• A series of labeled DNA molecules are generated,

each terminated by the dideoxynucleotide in each reaction

• These fragments of DNA are then separated

according to size by gel electrophoresis and detected by exposure of the gel to X-ray film

• The size of each fragment is determined by its

terminal dideoxynucleotide, so the DNA sequence corresponds to the order of fragments read from the gel

90

91

92

93

Fluorescence-based DNA sequencing

• Large-scale DNA sequencing is frequently performed

using automated systems using fluorescence-based reactions using labeled ddNTPs

• In this case, all four terminators can then be placed

in a single tube, and only one reaction is necessary

• The reactions are run into one lane on a gel and a

machine is used to scan the lane with a laser

94

95

Detection of fragments

• The wavelength of fluorescence can be interpreted

by the machine as an indication of which reaction (ddG, ddA, ddT, or ddC) the product came from

• The fluorescence output is stored in the form of

chromatograms

96

97

Why is fluorescence better than radioactivity?

• It eliminates the use of radioactive reagents
• Can be readily automated

98

Polymerase Chain Reaction

• In 1984, Kary Mullis devised a method called the polymerase

chain reaction (PCR) for amplifying specific DNA sequences

• PCR allows the DNA from a selected region of a genome to be

amplified a billionfold, effectively "purifying" this DNA away from the remainder of the genome

• The PCR method is extremely sensitive; it can detect a single

DNA molecule in a sample

99

100

Components of PCR reaction

• A pair of primers that hybridize to the target DNA.

– These primers should be specific for the target sequence and which are often about 15-25 nucleotides long. The region between the primers is amplified

• All four deoxyribonucleoside triphosphates (dNTPs:

dATP, dCTP, dGTP and dTT)

• A heat-stable DNA polymerase

101

DNA polymerases

• Suitably heat-stable DNA polymerases have been
• btained from microorganisms whose natural habitat

is hot springs

• For example, the widely used Taq DNA polymerase is
• btained from a thermophilic bacterium, Thermus

aquaticus, and is thermostable up to 94°C

102

PCR cycle

• Denaturation, typically at about 93-95°C. At this

temperature the hydrogen bonds that hold together the two polynucleotides of the double helix are broken, so the target DNA becomes denatured into single-stranded molecules

• Reannealing at temperatures usually from about 50°C

to 70°C where the primers anneal to the DNA

• DNA synthesis, typically at about 70-75°C, the
• ptimum for Taq polymerase

103

104

105

106

107

108

PCR cycles

• 20-30 cycles of reaction are required for DNA amplification,

– the products of each cycle serving as the DNA templates for the next-hence the term polymerase "chain reaction“

• Every cycle doubles the amount of DNA
• After 30 cycles, there will be over 250 million short products

derived from each starting molecule

109

Detection of DNA fragments

• This DNA fragment can be

easily visualized as a discrete band of a specific size by agarose gel electrophoresis

110

Importance of primers

• The specificity of

amplification depends on the specificity of the primers to not recognize and bind to sequences

• ther than the intended

target DNA sequences

• How can you prevent it?
• How can you take

advantage of it?

Annealing temperature

111

Disadvantage of PCR

• Primers must be known
• Contamination
• Product length is limited (usually <5 Kb)
• Accuracy is an issue
• Not quantitative

112

What are the strengths of PCR?

• Easy, fast, sensitive, robust
• Discovery of gene families
• Disease diagnosis

113

Forensic medicine

• An individual DNA profile is highly distinctive because

many genetic loci are highly variable within a population

• PCR amplification of multiple genes is being used to

establish paternity and criminal cases

114

Quantitative PCR (qPCR)

• Measurement of amount of DNA in a sample

115

• http://www.youtube.com/watch?v=kvQWKcMdyS4
• http://www.bio.davidson.edu/courses/immunology/flas

h/rt_pcr.html

Components of the human genome

116

Repetitive DNA sequences

117

Tandem vs. dispersed

118

Satellite (macro-satellite) DNA

• Repeats of 100 to 6500 bp
• Tandemly centromeric repeats (171 bp)

unique to each chromosome

» Each chromosome has its unique sequence » possible to make DNA probes specific to each

• Telomeric repeats

119

VNTRs (minisatellite)

• Mini satellite sequences or VNTRs (variable number
• f tandem repeats)
• They are composed of 20 to 100 bp repeats

120

STRs (microsatellites)

• STRs (short tandem repeats) composed of 2 to 10 bp

repeats

121

Polymorphisms of VNTR and STR

• The number of repeats in micro and mini satellites

are highly variable (polymorphic)

• Useful in:
• Gene mapping
• DNA profiling for paternity testing, forensic testing,

confirmation of relatedness and dead body identification

122

The bad side of STRs

• Reduction or expansion of STR can be pathogenic
• Unstable expansion of short tandem repeats is

characterised by anticipation

• Disease associated with large expansions outside

coding sequences

• Myotonic dystrophy (DM1)
• Friedrich ataxia (FA)
• Disease associated with modestexpansions outside

coding sequences

– Huntington disease (HD)

123

PCR of VNTRs

• VNTR blocks can be extracted and analyzed by RFLP or PCR

and size determined by electrophoresis

124

VNTR in medicine and more

• The picture below illustrates VNTR allelic length

variation among 6 individuals.

The likelihood of 2 unrelated individuals having same allelic pattern extremely improbable

125

126

127

SINEs and LINEs

• Two thirds (66.7%) of the repetitive non-coding DNA

sequences

• Dispersed throughout the genome
• Divided into short and long interspersed sequences,

SINES and LINES

128

LINEs (Long interspersed elements)

• 7000 bp in length
• Represent about 4% of our total human genome

129

SINEs (short interspersed elements)

• Shorter interspersed elements 90 to 500 bp in length

– Example: Alu sequence (~300 bp)

• Alu sequences are unique to humans (and some apes)
• 10% of the total human genome

130

Transposons

• SINEs and LINEs are transposable (jumping or moving)

elements

• Their origin is retroviruses

131

Effect of trasnposons

132

Pathogenic transpososns

• Transposable elements

can cause mutations (Hemophilia) and gene rearrangements and duplication (e.g., beta globin genes)

• Transposons are

mutagens

133

Single nucleotide polymorphism (SNPs)

• Another source of genetic variation
• Single-nucleotide substitutions of one base for

another

• Two or more versions of a sequence must each be

present in at least one percent of the general population

• SNPs occur throughout the human genome - about
• ne in every 300 nucleotide base pairs.

– ~10 million SNPs within the 3-billion-nucleotide human genome

134

Categories of SNPs

135