Lecture 6- DNA repair

Question 1

Give some example of how DNA molecules can be damaged

Answer 1

1. Thermal degradation
2. Metabolic byproducts/oxidation
3. Environmental substances
4. Radiation

Question 2

Which bases are purines and which are pyrimidines?

Answer 2

G and A = purines

T and C = pyrimidines

Question 3

Describe the base pairings

Answer 3

• A pairs with T (2 hydrogen bonds)

* C pairs with G (3 hydrogen bonds so are more thermally stable)

Question 4

Which group on thymine doesn’t hydrogen bond?

Answer 4

Methyl group

Question 5

What happens to cytosine in the presence of water?

Answer 5

• deamination
• loses amino group and converted to carbonyl group
• looses NH3
• causes cytosine to be converted to uracil which then hydrogens bonds with adenine

Question 6

Name 3 ways DNA bases can be damaged

Answer 6

1. Deamination of cytosine
2. Oxidative damage
3. Hydrolytic attacks

Question 7

What is a transition mutation?

Answer 7

refers to a point mutation that changes a purine nucleotide to another purine, or a pyrimidine nucleotide to another pyrimidine.

Question 8

Why are transition mutations more likely to occur than transversions?

Answer 8

Substituting a double ring structure for another double ring structure is more like than substituting a single ring for a double ring (and vice versa)

Question 9

What is a transversion mutation?

Answer 9

Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa

Question 10

What is a frameshift mutation?

Answer 10

Frameshift mutation is a type of mutation involving the insertion or deletion of a nucleotide in which the number of deleted base pairs is not divisible by three. If a mutation disrupts this reading frame, then the entire DNA sequence following the mutation will be read incorrectly

Question 11

What is photolytic conversion?

Answer 11

Light energy modifying the chemistry of the carbon-carbon double bond

Question 12

How is depurination resolved?

Answer 12

By the BER pathway

Question 13

Describe the BER pathway

Answer 13

1. Uracil DNA glycosylase remove particular base (deaminated cytosine) leaving DNA helix with a missing base
2. AP endonuclease and phosphodiesterase remove sugar phosphate creating a single-nucleotide gap
3. DNA polymerase adds/replaces nucleotide into the gap so the U is restored to a C
4. DNA ligase seals the nick

Question 14

How do BER glycosylases identify errors?

Answer 14

Using base-flipping strategy

Question 15

Describe the nucleotide excision repair pathway

Answer 15

1. Excision nuclease cuts out the region around the pyrimidine dimer, removing the damaged section of DNA
2. DNA helicase helps with this and unwinds the DNA into single strands
3. Left with DNA helix with 12-nucleotide gap
4. DNA polymerase and DNA ligase then add nucleotides and seal the gap by using the template strand

Question 16

How are pyrimidine dimers removed?

Answer 16

By the nucleotide/short patch excision repair pathway

Question 17

Describe the trans-lesional DNA synthesis pathway

Answer 17

1. Covalent modifications occur to sliding clamp when polymerase encounters DNA damage
2. This causes the DNA polymerase to be released
3. The sliding clamp then allows the loading of translesion polymerase by assembly factors
4. Translesion DNA polymerase then does some DNA synthesis to ‘mend’ the damaged DNA

Question 18

What are the 2 mechanisms to repair double stranded breaks?

Answer 18

1. Nonhomologous end joining

2. Homologous recombination

Question 19

Why are double strand breaks particularly hazardous and what causes them?

Answer 19

No template strand to facilitate repair

Caused by: ionising radiation, replication errors and oxygen radicles

Question 20

How does nonhomologous end joining repair double stranded DNA breaks?

Answer 20

1. The ends of the DNA break are recognised by Ku heterodimers and can be processed and made shorter
2. The ends are then joined together by ligation and repaired
3. Can cause a deletion of part of the DNA sequence which can lead to a missense mutation
4. Also required DNA-PK and ATM protein kinases function

Question 21

How does homologous recombination repair double stranded DNA breaks?

Answer 21

1. Processing of 5’ ends by nuclease at the point of the DNA break to create sticky ends
2. Strand exchange occurs by complementary base pairing
3. Using the information from the sister chromatids homologous recombination occurs
4. Invalid strand is released/broken and the double helix reforms
5. DNA synthesis continues using strand from damaged DNA as template to fill gaps from the break
6. DNA ligation
7. Damaged repaired accurately using sister chromatid as a template

Question 22

What are the steps to perform a biochemical analysis of complementation groups?

Answer 22

1. Make cell lysates
2. Use artificially damage DNA templates
3. Use repair assay to determine enzymatic function for each group
4. Repair the transcribed strand is faster than the non-transcribed strand

Question 23

What does trans-lesional DNA polymerase lack and cause when performing trans-lesional DNA synthesis?

Answer 23

LACK: precision in template recognition and substrate base choice and exonucleolytic proof reading activity.

CAUSE: most base substitutions and single nucleotide deletion mutations