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

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