DNA Damage and Repair

Question 1

What is deamination and give examples

Answer 1

Primary amino groups of nucleic acid bases can be converted to ketogroups (C->U)
Adenine -> hypoxanthine
Guanine -> xanthine
5-methyl cytosine -> thymine

Question 2

What is chemical modification

Answer 2

Nucleic acid bases are susceptible to modifications by a wide variety of chemical agents
Common for Adduct formation
Several types of hyper-reactive oxygen (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) can modify DNA bases

Question 3

What is a common product of thymine oxidation

Answer 3

Thymine glycol

Question 4

How can hyper-reactive oxygen species be generated

Answer 4

As byproducts during normal oxidative metabolism
Ionising radiation (x-rays, gamma rays)

Question 5

How do environmental chemicals modify DNA bases

Answer 5

Addition of methyl or alkyl groups

Question 6

What is photodamage

Answer 6

Ultraviolet light is absorbed by the nucleic acid bases

The resulting influx of energy can induce chemical changes

Question 7

What are the most frequent photoproducts

Answer 7

Consequences of bond formation between adjacent pyrimidines within one strand.

Question 8

What are the types of DNA damage

Answer 8

Nick
Gap
Thymine dimer
Base pair mismatch

Question 9

What carcinogens can cause to damage DNA

Answer 9

dietary
lifestyle
environmental
occupational
medical
endogenous

Question 10

Which types of radiation can damage DNA

Answer 10

ionizing
solar
cosmic

Question 11

Describe the DNA damage by carcinogens

Answer 11

DNA adducts and alkylation
Base hydroxylations
Base dimers and chemical cross-links
Double and single strand breaks

Question 12

Describe the phases of metabolism

Answer 12

Phase I - addition of functional groups
Mainly cytochrome P450-mediated

Phase II - Conjugation of phase I functional groups 
Generates polar (water soluble) metabolites

Question 13

Give examples of Phase I reactions

Answer 13

Oxidation
Reduction
Hydrolysis

Question 14

Give examples of phase II reactions

Answer 14

Sulphation
Glucuronidation 
Acetylation
Methylation
Amino acid 
Glutathione conjugation

Question 15

Where can polycyclic aromatic hydrocarbons, aflatoxin B1 and 2-napthylamine be found

Answer 15

Polycyclic hydrocarbons - Formed from combustion of fossil fuels and tobacco

Aflatoxin B1 - grains and peanuts

2-napthylamine - Dye-stuffs (past)

Question 16

Describe the epoxidation of B[a]P

Answer 16

2 steps
B[a]P is converted to DNA adducts using liver P450 and epoxide hydrolase
DNA adducts attaches itself to bases and causes mutations

Question 17

Describe the epoxidation of aflatoxin B1

Answer 17

Conversion first by cytochrome p450 oxidase into a very active form.
Reaction with guanine on DNA (N7 position) to cause adduct formation

Question 18

Describe the metabolism of 2-naphthylamine

Answer 18

First conversion by cytochrome p450 oxidase (CYP1A2)
Second is a phase II reaction using glucuronyl transferase
pH of the urine causes conversion to a DNA-reactive electrophile that causes bladder tumours

Question 19

What is an adduct

Answer 19

Segment of DNA bound to a cancer-causing chemical

Often from the conversion of a small molecule to a much larger one

Question 20

Explain how UV radiation is able to damage DNA

Answer 20

Pyrimidine (thymine) dimers

Skin cancer

Question 21

Explain how ionising radiation is able to damage DNA

Answer 21

Generates free radicals in cells
Includes oxygen free radicals
super oxide radical (O2•)
hydroxyl radical (HO•)

Possess unpaired electrons
electrophilic and therefore seek out electron-rich DNA

Question 22

Describe the oxygen free radical attack on DNA

Answer 22

Double and single strand breaks
Apurinic + apyrimidinic sites (reactive)
Base modifications

Question 23

What are the base modifications that oxygen free radicals can cause

Answer 23

ring-opened guanine +adenine
thymine + cytosine glycols
8-hydroxyadenine + 8-hydroxyguanine (mutagenic)

Question 24

What is the role of p53 in dealing with cellular stress

Answer 24

(tumour suppressor gene)
Responds to cellular insults:
Mitotic apparatus dysfunction 
DNA replication stress
Double-Strand breaks

MDM2 keeps p53 inactive. It is then lost in damage, allowing p53 to activate transcriptional pathways, including DNA repair

Question 25

What are the types of DNA repair

Answer 25

Direct reversal of DNA damage Base excision repair (mainly for apurinic/apyrimidinic damage) Nucleotide excision repair (mainly for bulky DNA adducts) During or post replication repair

Question 26

Describe direct DNA repair

Answer 26

the reversal or simple removal of the damage by the use of proteins which carry out specific enzymatic reactions

Question 27

What is the actions of MGMT and photolyases

Answer 27

directly reverses some simple alkylation adducts Photolyases repair thymine dimers

Question 28

How are DNA mismatches repaired

Answer 28

Involves scrutinisation of DNA for apposed bases that do not pair properly Mismatches that arise in replication are corrected via proof-reading (comparison of old and new) - preference for the new strand Other systems deal with mismatches generated by base conversions, such as those which result from deamination

Question 29

Describe base excision repair of DNA damage

Answer 29

Damage to base (but not to the phosphodiester backbone) 1. DNA glycosylase removes the base of the nucleotide without affecting the backbone 2. AP-endonuclease cuts the DNA strand open 3. DNA polymerase adds the correct base 4. DNA ligase closes the strand

Question 30

Describe the nucleotide excision repair of DNA damage

Answer 30

1. Endonuclease removes the phosphodiester bond 2. Helicase removes a large chunk of bases (including backbone) 3. DNA polymerase remakes the strand 4. DNA ligase closes strand

Question 31

Give examples of human genetic disease involving nucleotide excision repair

Answer 31

Xeroderma pigmentosum Trichothiodystrophy Cockayne's syndrome

Question 32

When are double-strand breaks made

Answer 32

Under physiological conditions during somatic recombination and transposition. e.g. V(D)J recombination During Homologous Recombination. As a result of ionizing radiation and oxidative stress induced DNA damage.

Question 33

Describe DNA double strand break repair

Answer 33

Direct joining of the broken ends. This requires proteins that recognize and bind to the exposed ends and bring them together for ligating. Non-complementary nucleotides - Nonhomologous End-Joining (NHEJ).

Question 34

What are the consequences of incorrectly repaired carcinogen damage leading to altered DNA

Answer 34

DNA replication and cell division -> fixed mutation Transcription/translational gives aberrant proteins Carcinogenesis if the targets are mutated Apoptosis and cell death

Question 35

What are the types of therapeutic agents used to cause DNA damage in tumour cells

Answer 35

Alkylating agents Agents that make bulky adducts Agents that induce double strand breaks

Question 36

How is DNA damage tested for

Answer 36

1. Structural alerts/SAR 2. In vitro bacterial gene mutation assay 3. in vitro mammalian cell assay 4. in vivo mammalian assay 5. Investigation in vivo mammalian assays

Question 37

Describe the Ames test for mutagenicity of chemicals

Answer 37

The chemical tested is added to rate liver enzyme preparations (S9) and bacteria that do not synthesise histidine Conversion of chemical to reactive metabolite On histidine-free media: if mutations occur in bacterial genome then bacteria acquire ability to synthesise histidine = colonies

Question 38

Give an example of a bacterium that does not synthesise histidine

Answer 38

Salmonella

Question 39

How are chromosomal aberrations detected

Answer 39

Treat mammalian cells with chemical in presence of liver S9. Look for chromosomal damage

Question 40

Describe the in vitro micronucleus assay

Answer 40

Cells treated with chemical and allowed to divide Binucleate cells assessed for presence of micronuclei Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss)

Question 41

Describe the murine bone marrow micronucleus assay

Answer 41

Treat animals with chemical and examine bone marrow cells or peripheral blood erythrocytes for micronuclei