DNA Repair

Question 1

What are the 2 main types of DNA damage?

Answer 1

Chemical alteration and spontaneous damage.

Question 2

What is meant by exogenous chemical alteration?

Answer 2

Damage caused by environmental mutagens such as UV radiation.

Question 3

What is meant by endogenous chemical alteration?

Answer 3

Damage caused by internally generated mutagens such as hydroxyl radicals which as formed as by products of metabolic reactions.

Question 4

Give examples of spontaneous damage to DNA.

Answer 4

Deamination and depurination.

Question 5

How does UV radiation damage DNA?

Answer 5

Induces formation of pyrimidine dimers where two adjacent pyrimidines are joined by a cyclone take ring. Causes loss of aligned C=C bonds, loss of planarity and distortion of the DNA structure.

Question 6

How does alkylation damage DNA?

Answer 6

Addition of methyl/ethyl groups to various positions on the DNA bases.

Question 7

How do carcinogens damage DNA?

Answer 7

React with DNA bases resulting in the addition of large bulky chemical groups to the DNA molecule. Many carcinogens are activated via reactions with cytochrome P450, this means that non-toxic drugs may become toxic once processed.

Question 8

How often does depurination of A/C/G occur?

Answer 8

Approximately 500 times a day.

Question 9

What occurs as a result of depurination of A/C/G?

Answer 9

Changes base pair properties and causes mismatches.

Question 10

How often does depurination of A/G occur?

Answer 10

Approximately 18,000 times a day.

Question 11

What occurs as a result of depurination of A/G?

Answer 11

Cleavage of the bond between the purine base and deoxyribose- elimination of the base.

Question 12

Which is more damaging deamination or depurination?

Answer 12

Depurination- is more toxic and more mutagenic.

Question 13

What are the two main methods of DNA repair? And which is used most often.

Answer 13

Direct reversal- rare, excision repair- more likely.

Question 14

How are pyrimidines dimers repaired?

Answer 14

Visible light is used by a photo reacting enzyme to break the cyclobutane.

Question 15

Where is pyrimidine dimer direct reversal seen?

Answer 15

In e.coli, yeast and some plants.

Question 16

How is guanine methylation repaired?

Answer 16

O6-methylguanine methyltransferase has a Cys residue that is methylated by the methylguanine. This is a suicide enzyme.

Question 17

What occurs during excision repair?

Answer 17

Damaged bases are removed and replaced with newly synthesised DNA.

Question 18

Describe base-excision repair.

Answer 18

DNA glycosylase cleaves glycosidic bond between base and deoxyribose. Leaves an AP site and the backbone intact. AP endonuclease cleaves DNA chain. Deoxyribosephosphodiesterase removes remaining deoxyribose. Gap filled by DNA Pol and sealed by ligase.

Question 19

Describe nucleotide excision repair.

Answer 19

3’ and 5’ endonucleases cleave the backbone upstream and downstream of a bulge cause by a thymine dimer. Helicase unwinds DNA, causing excision of a 30nt oligonucleotide containing the thymine dimer. Gap is filled by DNA Pol and sealed by ligase.

Question 20

What enzymes catalyse nucleotide excision repair in E.coli and what do mutations in these cause?

Answer 20

The uvrABC gene productions. Mutations lead to high UV sensitivity.

Question 21

What enzymes catalyse nucleotide excision repair in eukaryotes and what do mutations cause?

Answer 21

The RAD gene products. Mutations cause Xeroderma pigmentosa.

Question 22

Describe Xeroderma pigmentosa.

Answer 22

Individuals cannot repair DNA damage caused by UV exposure. RARE genetic disease. Results in reduced life expectancy due to high levels of skin cancer if exposed to UV light. Sufferers must be completely shielded from UV radiation.

Question 23

When is the mismatch repair mechanism used?

Answer 23

To repair post-replication mismatches.

Question 24

Which is the only repair mechanism to increase fidelity during DNA replication?

Answer 24

Mismatch repair

Question 25

What proteins are involved in mismatch repair in E.coli?

Answer 25

MutHLS

Question 26

What is the role of MutS?

Answer 26

Recognises mismatch

Question 27

What is the role of MutL?

Answer 27

Binds MutS at mismatches and recognises hemimethylated dam sites. Uses ATPase activity to translocate along the DNA looking for a hemimethylated dam site.

Question 28

Why is DNA repair a crucial process?

Answer 28

Information must be passed on from one generation to the next and must be stable over many lifetimes. DNA must be repaired to limit information loss.

Question 29

What is the role of MutH?

Answer 29

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Question 30

Describe the process of mismatch repair in E.coli.

Answer 30

MutS recognises mismatch and MutL binds. MutH is activated when bound to MutL and cleaves the non-methylated strand opposite the hemimethylated dam site. This defines the newly synthesised strand for repair. UvrD helicase excises everything between the mismatch site and the excision site. Gap filled by DNA Pol III and sealed by ligase.

Question 31

Where is the nick produced by MutH?

Answer 31

Can be upstream or downstream of the mismatch- affects which endonuclease is needed for excision.

Question 32

What catalyses mismatch repair in mammalian cells?

Answer 32

MSH complex.

Question 33

How are daughter strands distinguished from parental strands in mammalian cells?

Answer 33

Excision occurs between Okazaki fragments and the mismatch site.

Question 34

Which part of the replication machinery remains bound to the DNA during mismatch repair in mammalian cells?

Answer 34

beta clamps- remain bound and interact with repair proteins.

Question 35

What can be the result of mutations in the MSH complex?

Answer 35

Non-polyposis colorectal cancer.