Xeroderma Pigmentosum
Jan 02, 2016
Xeroderma Pigmentosum
Study Guide/Outline--Mutations
Mutation Mechanisms• A gene may have a mutation rate of “1.4 x10-5” What exactly does
this number mean? (from class)• What are the molecular mechanisms by which mutations arise in
the DNA? What can happen during DNA replication? Recombination, chemically?
• What is the difference between transitions and transversions?
Effects on Protein/Effects on the Organism• What are the differences between a missense, nonsense, and
frameshift mutation? (and how do they arise)? Why does a silent mutation not result in an amino acid change?
• Mutations in DNA sequence may be written as “T352C”, while mutations in amino acid sequence may be written as “Met 54 Val”. What is meant by this nomenclature?
• The effect of a mutation may be reversed in an organism, either a true reversion at the same nucleotide, or through second mutations. Explain the difference between a true reversion, partial reversion, and suppressor mutations (intragenic or intergenic).
• What is the difference between a somatic and germline mutation (including passing on mutation to offspring and what proportion of cells in the organism are mutant)?
Different mutation rates for different genes
Disease Locus or Gene Mutation Rate
Achondroplasia (dominant dwarfism)
FGF-R3 (fibroblast growth factor receptor 3)
0.6 – 1.4 x 10-5
Duchenne Muscular Dystrophy
DMD 3.5 – 4.5 x 10-5
Hemophilia A Clotting Factor VIII 3.2 – 5.7 x 10-5
Types of Mutations
Protein Changing--Deleterious or neutral (sometimes beneficial) mutations
•Missense--a.a. different a.a. •Sometimes neutral effect on protein if new a.a. is chemically similar to old
•Nonsense—a.a. codon stop codon (truncation of protein)•Insertion or deletion of nucleotideshift in reading frame (frameshift mutation missense then stop codon)
Non protein-changing•Silent mutations (non-a.a. changing)--neutral Mutations
Changes in expression pattern• Mutations in the promoter or regulatory
regions• position effect
The genetic code
Frameshift Mutations
Frameshift U G C A AA U G
Met
A A G
Lys
G C G
Ala
C A UU U
U
G
Leu
Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and
shortened protein
mRNA
Protein
NormalmRNA
Protein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
Frameshift Mutations
Frameshift U G C A AA U G
Met
A A G
Lys
G C G
Ala
C A UU U
U
G
Leu
Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and
shortened protein
mRNA
Protein
NormalmRNA
Protein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
(a) Position effect due to regulatory sequences
(b) Position effect due to translocation to a heterochromatic chromosome
Codingsequence
Corepromoter
Regulatorysequence
Gene B
Codingsequence
Corepromoter
Gene A
Inversion
Translocation
Core promoter for gene A ismoved next to regulatorysequence of gene B.
Activegene
Heterochromaticchromosome(more compacted)
Euchromaticchromosome
Shortened euchromaticchromosome
Geneis nowinactive.
Translocatedheterochromaticchromosome
B
A
B
A
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Brooker, Fig 18.2a and b
Mutations causing changes in gene
expression
Achondroplasia
Mutation in Fibroblast Growth Factor Receptor 3 (FGFR-3)
Chromosome 4p16.3
Almost all casesGly 380 Arg
Nonsense and Frameshift Mutations in APC gene cause Familial Adenomatous Polyposis
• Inner colon epithelia is covered in polyps
• Risk of extracolonic tumors (upper GI, desmoid, osteoma, thyroid, brain, other)
• Untreated polyposis leads to 100% risk of cancer
• Prevention—prophylactic colectomy
Gel Electrophoresis to detect truncated APC proteins in FAP families
• DNA transcribed to mRNA
• RNA translated to protein
• Protein run on gel
• Truncated protein has different mobility in gel
DNA
mRNA
Protein
Gel
Normal Mutated
What will the protein bands look like on the gel?
Gel Electrophoresis to detect truncated APC proteins in FAP families
• DNA transcribed to mRNA
• RNA translated to protein
• Protein run on gel
• Truncated protein has different mobility in gel
DNA
mRNA
Protein
Gel
Normal Mutated
Shorter mutant protein
runs faster
Germ-linemutation Gametes
Embryo
Matureindividual
Mutation isfoundthroughoutthe entirebody.
Half ofthe gametescarry themutation.
Somaticmutation
Patch ofaffectedarea
None ofthe gametescarry themutation.
(a) Germ-line mutation (b) Somatic cell mutation
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Figure 18.4a and b
The earlier the mutation, the
larger the patch
Different results of somatic vs. germline mutations
Sources of mutation
• Mistakes in DNA replication:– Mismatch pairing due to “wobble-like” pairing– Slippage of DNA polymerase at repeated sequences– Tri-nucleotide repeat expansion (e.g. Huntington's gene,
FRAXA. See fig 18.12)
• Spontaneous mutations:– Depurination – De-amination– Tautomeric shift (see fig 18.10)
• Oxidative stress and ROS• Mutagen inducers
– Chemical mutagens: ethidium bromide, 5-BrdU– Ionizing radiation– UV radiation
Fig 17-13 Wobble base pairing leads to a replicated errorWobble base pairing leads to a replicated error
Insertions and Deletions may result from strand slippage
Insertion in newly synthesized strand
Deletion in newly synthesized strand
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• In normal individuals, trinucleotide sequences are transmitted from parent to offspring without mutation– However, in persons with TRNE disorders, the length of a trinucleotide repeat
increases above a certain critical size
• It also becomes prone to frequent expansion
• This phenomenon is shown here with the trinucleotide repeat CAG
CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG
CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG
n = 11
n = 18
18 - 48
Fig-18.12
(a) Formation of a hairpin with a trinucleotide (CTG) repeat sequence
One DNA strand with a trinucleotide repeat sequence
One DNA template strand prior to DNA replication One DNA template strand prior to DNA replication
Trinucleotide (CTG) repeat
TNRE TNRE
Hairpin with CG base pairing
C C T GT G C T G C T G C T G C T GT TT A A G C AG C C A A G T TCC AT A
Hairpin formation
CT
G
CT
GC
TG
CT
C
TG
CT
G
T TT A A G C AG C C A A G T TCC AT A
(b) Mechanism of trinucleotide repeat expansion (c) Mechanism of trinucleotide repeat deletion
DNA replication beginsand goes just past the TNRE. Hairpin forms in template strand
prior to DNA replication.
DNA replication occurs andDNA polymerase slips overthe hairpin.
DNA repair occurs.
DNA polymerase slips offthe template strand and ahairpin forms.
DNA polymerase resumesDNA replication.
DNA repair occurs.
OR OR
DNApolymerase
TNRE is the same length. TNRE is the same length.
TNRE is longer. TNRE is shorter.
Expansion of tri-nucleotide
repeats
Brooks, Fig 18.13
Uracil
HNO2
HNO2
Adenine
Cytosine
H
H
Sugar
Sugar
Sugar
O
O
H
Sugar
NH
H
NH
H
Template strand After replication
Hypoxanthine HN
H
H
O
O
H
H
Cytosine
Sugar
NH2
O
H
H
Adenine
H
NH NH2
Sugar
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Figure 18.9a 18 - 35
UracilCytosine
O
(a) Deamination of cytosine
+ NH3+ H2O
O
H H
H
NH2
O
H
H
SugarSugar
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N
N
N
N
Figure 18.9b 18 - 38
Thymine
O
H
H
O
CH3CH3
+ NH3
5-methylcytosine
(b) Deamination of 5-methylcytosine
+ H2O
NH2
O H
SugarSugar
N
NN
N
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H
N
O
N
CH3
Thymine
Cytosine
NH2
O
H
H
N
NH
O
H
H
NH
Keto form Enol form
Sugar
Sugar Sugar
N H
N
OH
CH3
O
Tautomeric shift
Tautomeric shift
Amino form Imino form
Common Rare
(a) Tautomeric shifts that occur in the 4 bases found in DNA
O
H
N
Sugar
N
N
N
N N
N
N
N
N
H2N N
N
H
N
Adenine
Guanine
O
H N
N
H
H2N
H
N
N
N
N
H
N
NH
OH
Sugar
H N
N
H
N
N
N
Sugar
Keto form Enol form
Tautomeric shift
Amino form Imino form
Common Rare
N NH
Sugar Sugar
Tautomeric shift
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N N
Figure 18.10a
Co
mm
on R
are
Tautomeric shifts of nucleotides
change the pairing properties
Figure 18.10b
Cytosine (imino) Adenine (amino)
H
N
N H
N
NSugar
NO
H
SugarN
NN
H
H
H
N
N H
H
N
O
SugarN
H
OH3C
OSugar
Thymine (enol) Guanine (keto)
H
HH
NNN N N
N
NN
N
NN
Mis–base pairing due to tautomeric shifts
(c) Tautomeric shifts and DNA replication can cause mutation.
5′ 3′
3′ 5′
T A
5′ 3′
3′ 5′
T A
5′ 3′
3′ 5′
5′ 3′
3′ 5′
T A
5′ 3′
3′ 5′
T A
5′ 3′
3′ 5′
T G
5′ 3′
3′ 5′
T A
A thymine baseundergoes atautomeric shift priorto DNA replication.
Basemismatch
A second roundof DNA replicationoccurs.
DNA molecules foundin 4 daughter cells
MutationC G
Temporary tautomeric shift
Shifted back to its normal form
Figure 18.10c
Depurination produces a “gap” in the DNA sequence
Deamination of cytosine bases results in C T Transition
Unequal crossing over produces insertions and deletions
5-BrdU is an unstable chemical analog of Thymine
N
5-bromouracil (keto form)
Adenine
Sugar
Sugar
5-bromouracil (enol form)
Guanine
Sugar
Sugar
(a) Base pairing of 5BU with adenine or guanine
H
HH
O
O N
O
Br
H
H
Br H
O
O
H
H
H
H
N
N
N
N
NN
N
N
N
N
NN
Figure 18.14a
Brooker, Fig 18.11
GuanineBase pairs with cytosine
8-oxoguanine(8-oxoG)
Base pairs with adenine
H
43
2
156
7
89
O
NH2
HROS
H
H
H
43
2
156
7
89
O
NH2
H
O
N
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NN
NN
NN
N
Oxidative stress and DNA damage causes G to T transversions
• Ionizing radiation – Includes X-rays and gamma rays– Has short wavelength and high energy– Can penetrate deeply into biological materials– Creates chemically reactive molecules termed
free radicals– Can cause
• Base deletions• Single nicks in DNA strands• Cross-linking• Oxidized bases• Chromosomal breaks
18 - 62
• Nonionizing radiation
– Includes UV light
– Has less energy
– Cannot penetrate deeply into biological molecules material
– Causes the formation of cross-linked thymine dimers
– Thymine dimers may cause mutations when that DNA strand is replicated
– Refer to Figure 18.15
18 - 63
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Figure 18.1518 - 64
Ultravioletlight
Thymine dimer
Thymine
Thymine
HH
H
O
H
H
O
CH3
CH2
HH
H
OH
H O
CH3
CH2
–
–
HH
H
O
H
H
O
CH3
CH2
HH
H
OH
H O
CH3
CH2
–
–
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HH
OOO
O
P
N
N
HH
OOO P
N NO
O
HH
OOO P
N
N
HH
OOO P
N NO
O
O
O
O
Go over lecture outline at end of lecture