Gene mutation and DNA repair

Question 1

Variation by mutation

Answer 1

A change in composition of bases in the DNA
(genetic variation)

• Beneficial mutations are usually passed to future generations and become distributed in the population.
• Detrimental mutations are usually not passed on as they result in abnormal/infertile/dead organism.

This form the basis of evolution.

Question 2

Gene mutation

Answer 2

Failure to store genetic information faithfully

• The genetic code consists of triplet codons (three nucleotides specifies a single amino acid in the corresponding polypeptide).
• Any change that disrupts these sequences or the coded information provides sufficient basis for a mutation.

Question 3

2 types of targets

Answer 3

Somatic mutation and gametic mutations

Question 4

Somatic mutations

Answer 4

Affects one individual and are not transmitted to future

generations

Question 5

Gametic mutations

Answer 5

Part of germline (i.e. sperm or egg) so transmitted to

offspring and enters the gene pool (i.e. effects subsequent generations)

Question 6

List the 2 types of mutations

Answer 6

Point mutations and frameshift mutations

Question 7

Point mutations

Answer 7

• Involve the substitution / replacement of a base
• Protein length remains the same but the changed base may or may not lead to a change in the amino acid due to the redundancy of the genetic code

Question 8

Frameshift mutations

Answer 8

Result in a change of the transcript and translated protein due to a change in the reading frame

Question 9

Missense mutation

Answer 9

Point mutation that induces single base substitution and change the corresponding amino acid.

Question 10

Nonsense mutation

Answer 10

Point mutation that induces stop codon in the open reading frame.

Question 11

Silent mutation

Answer 11

Point mutation that induces single base substitution but it does not change the corresponding amino acid.

Question 12

Insert mutation

Answer 12

Addition of a base in the open reading frame that causes change in the triplet codon or reading frame.

Question 13

Deletion mutation

Answer 13

Deletion of a base in the open reading frame that causes change in the triplet codon or reading frame

Question 14

List the 2 classifications of mutations

Answer 14

1. spontaneous mutation

2. induced mutation

Question 15

Spontaneous mutation

Answer 15

• Caused by random changes in the nucleotide sequences of genes.
• These rare mutations arise as a result of errors during DNA replication.
• Once an error is present in the genetic code, it is reflected in the amino acid composition of the specified protein.
• If the changed amino acid is in a part of the protein that is critical to its structure or biochemical activity, then the protein becomes non-functional.

Question 16

3 characteristics of spontaneous mutation rates

Answer 16

• Is extremely low
• Varies considerably in different organisms
• Even within the same species, spontaneous mutation
  rate varies from gene to gene
• can be induced by mutagens

Question 17

Induced mutations

Answer 17

Caused by artificial factors which increases chemical

reactivity in cells leading to mutations.

Question 18

List 3 mutagens that can cause induced mutations

Answer 18

(a) High energy radiation
(b) Base analogues
(c) Other chemical mutagens

Question 19

UV light

Answer 19

• UV radiation from the sun produces thymine dimers
• The dimers distort DNA conformation and inhibit
  normal replication

Question 20

Ionization radiation from cosmic or mineral sources

Answer 20

• Gamma rays, X rays, cosmic rays etc
• Ionizing radiation is more energetic than UV radiation since it has a shorter wavelength
• Ionizing radiation produces DNA strand breaks

Question 21

Base analogues

Answer 21

• Molecules that substitute purines or pyrimidine during
  DNA replication
• Changes to the base analogue leads a new base pair to appear, this leads to point mutations
• Examples: 5-bromouracil (5-BU), 2-aminopurine

B-bromouracil (keto form) is just thymine with CH3 substituted with Br

Question 22

5-bromouracil

Answer 22

• > Acts as a thymine analogue
• > If 5-BU (keto form) is incorporated into DNA in place of thymine, it base-pairs with adenine
• > 5-BU then changes to the enol form which base-pairs with guanine during DNA replication
• After one round of replication, an AT pair is changed to GC pair
• 5-BU can cause a transition from AT pair to GC pair

Question 23

Acridine dyes

Answer 23

• 3 ringed molecules
• cause frameshift mutations
• addition or deletion of one or more base pairs in the sequence of the gene

Question 24

Examples of acridine dyes

Answer 24

Acridine orange, proflavin, acriflavine

Question 25

Mechanism of acridine dyes

Answer 25

-> Acridine dyes have roughly the same dimension as
nitrogenous bases

• > They intercalates between purines and pyrimidines of DNA
• > Intercalation of acridine dyes induces contortions in the DNA helix, causing deletions and additions

Question 26

UV induced damage

Answer 26

• UV radiation is mutagenic and causes formation of
  pyrimidine dimers in DNA, mainly between two adjacent thymine residues (i.e. thymine dimers).
• The thymine dimers distort the DNA conformation and block replication.

• This replication block is responsible for the UV killing
the cells.

Question 27

2 types of UV specific repair mechanisms

Answer 27

Light dependent repair (photoreactivation repair)

Light independent repair (Nucleotide excision repair)

Question 28

Photoreactivation repair

Answer 28

• Showed that UV-induced damage in E. coli could be partially reversed by exposure to visible light after irradiation
• Only blue light was necessary to correct radiation damage
• Further studies showed that this blue light effect is temperature sensitive (implying the involvement of a protein)
• This photoreactivation repair depended on a protein called photoreactivation (PR) enzyme

Question 29

3 steps in photoactivation repair

Answer 29

1) The PR enzyme binds specifically to a thymine dimer. Although the enzyme will associate with a dimer in the dark, it must absorb a photon of light to cleave the dimer
2) Once blue light is absorbed, the PR enzyme cleaves the bonds between thymine dimers to reverse the effect of UV radiation on DNA
3) The PR enzyme then dissociates from the DNA

Question 30

Nucleotide excision repair

Answer 30

-> Damaged DNA is removed and resynthesized using the opposite strand as template

• Isolated several E. coli mutants which showed increased sensitivity to UV light (i.e. mutants died faster than wild type when exposed to UV light)
• Four E. coli genes designated UVr (ultraviolet repair) were involved
• UvrA, UvrB, UvrC & UvrD are required for the excision of dimer

Question 31

3 steps in nucleotide excision repair

Answer 31

The thymine dimer is recognized and enzymatically cut out by UvrABCD.

This excision which includes several nucleotides adjacent to the dimer, leaving a gap in the DNA strand.

2) DNA polymerase I fills this gap by inserting nucleotides complementary to those on the intact strand. The enzyme adds these bases to the 3’ OH end
of the cut DNA.

3) The enzyme DNA ligase seals the final nick that remains at the 3’ OH end of the last base inserted, closing the gap.

Question 32

Nucleotide excision repair in humans

Answer 32

-> This type of mutation is detected by a complex of the XP-C (Xeroderma pigmentosum C) protein and 23B proteins.

-> The complex will recruit transcription factor TFIIH, whose helicase subunit (powered by ATP) will unwind and stabilize the helix until a bubble of 25
bases is formed.

• > Two endonucleases, XP-G and XP-F, will cut the unwanted bases, releasing it from the helix .
• > The gap is filled by DNA Polymerase d / e by inserting nucleotides complementary to those on the intact strand.
• > DNA ligase closes the nick.

Question 33

Xeroderma Pigmentosum (XP)

Answer 33

(Failure of Nucleotide Excision Repair)

Question 34

Xeroderma Pigmentosum (XP) cause

Answer 34

• > Rare autosomal recessive disorder in humans (very severe and may be lethal).
• > Predisposes individuals to epidermal pigment abnormalities.
• People with XP develop skin lesions when exposed to UV radiation (present in sunlight) and commonly develop skin cancer.
• Human XP cells are
• killed by smaller ultraviolet doses than cells from a normal person
• ability to remove thymine dimers from their DNA is very much reduced

• XP individuals lack nucleotide excision repair system which causes them to be susceptible to UV-induced skin damage.