DNA damage and repair

Question 1

Why are the nitrogenous bases so reactive

Answer 1

Carbon ring structures- allowing for potential of delocalisation of electrons
Double bonds- allowing for activation of these
Reactive side chains- just remove methyl group from thymine to make uracil

Generally, planar, cyclical-carbon structures are very reactive, and thus the bases are very reactive too.

Question 2

What are the potential outcomes of base modifications

Answer 2

Prevent replication

Cause mutations

Question 3

Describe deamination reactions

Answer 3

The primary amino groups of nucleic acid bases are somewhat unstable. They can be converted to ketogroups
NH2- =O (water in, ammonia out)
Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
Thousands of these occur daily in cells!

Question 4

Describe chemical modification

Answer 4

The nucleic acid bases are susceptible to numerous modifications by a wide variety of chemical agents. For example, several types of hyper-reactive oxygen (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) are generated as byproducts during normal oxidative metabolism.

All of these can modify DNA bases. A common product of thymine oxidation is thymine glycol:

Question 5

Describe the consequences of hyper-reactive oxygen species

Answer 5

Hyper-reactive oxygen species are also generated by ionizing radiation (X-rays, gamma rays).

Many environmental chemicals, including “natural” ones (frequently in the food we eat) can modify DNA bases, frequently by addition of a methyl or other alkyl group (alkylation). In addition, normal metabolism frequently leads to alkylation. Addition of larger molecules defines “adducts”.

These thymine glycols can take up these methyl groups and form adducts,

Question 6

Why is DNA the target for many carcinogens

Answer 6

Chemical carcinogens are usually metabolically activated and converted into electrophiles (they want electrons)

DNA is very electron rich

Question 7

What are the consequences of bulky DNA adducts

Answer 7

The electrophiles bind and form a covalent bond- bind covalently to DNA

The binding of these adducts causes problems, particularly during replication because it interferes with the ability of DNA polymerase to recognise the base (because of the bulky adduct)

Question 8

Describe photo damage to DNA

Answer 8

Ultraviolet light is absorbed by the nucleic acid bases, and the resulting influx of energy can induce chemical changes. The most frequent photoproducts are the consequences of bond formation between adjacent pyrimidines within one strand.
Intra-strand effect
Thymine dimers can form
These actually help protect the body from UV damage

Question 9

Describe the different types of DNA damage that can occur

Answer 9

Nick- break in phosphodiester bond as a result of ionising or radioactive damage
Gap- many nicks
Thymine dimers- disrupts topology of DNA double helix- which can act as a signal for DNA damage
Base pair mismatch- bulge in DNA double helix- no longer have complementary base pairing- can act as a signal for DNA damage.

Question 10

Describe some of the chemical carcinogens that can damage DNA

Answer 10

dietary
lifestyle- smoking 
environmental
occupational
medical
endogenous

Question 11

Describe how radiation can damage DNA

Answer 11

Radiation
ionizing
solar
cosmic

Question 12

Describe the importance of carcinogens

Answer 12

DNA damage can lead to mutation
Mutation may lead to cancer
Damaging DNA is an important strategy in cancer therapy - introduce them into the cells to make them unstable and impossible for them to survive.

Question 13

State some different types of DNA damage caused by carcinogens

Answer 13

DNA adducts
& alkylation
Base dimers &
chemical cross-links
Double & single
strand breaks
Base hydroxylations
& abasic sites formed- hydroxylation damages the base that much it's destroyed.

Question 14

Which group of chemicals can chemically modify and damage DNA

Answer 14

Polycyclic Aromatic Hydrocarbons
which are produced by the combustion of organic material, aflatoxins which are microbial bi-products that contaminate grain crops in hot and humid climates and aromatic amines that are commonly used industrial intermediates.
Common environmental pollutants
Formed from combustion of fossil fuels
Formed from combustion of tobacco

Question 15

Summarise the mammalian metabolism of carcinogens

Answer 15

§ Phase 1 – addition of functional groups – oxidations, reductions, hydrolysis- takes place in liver- convert hydrophilic carbon ring structures to something that can become more polar and thus excreted.

o Mediated mainly by cytochrome p450 enzymes.

§ Phase 2 – conjugation of phase 1 functional groups – glucuronidation, sulphation, glutathione conjugation, methylation, acetylation & amino-acid conjugation.

o Generates polar (water soluble) metabolites to excrete. Most carcinogens are insidious and only become carcinogenic after phase 1 metabolism.

Question 16

Where is benzo(a)pyrene BaP found

Answer 16

Coal tar
Cigarette smoke
Grilled meats
Involved in the carcinogenesis of lung and colorectal cancers.

Type 1 carcinogen- very potent

Question 17

Describe the two step epoxidation of Benzo(a)pyrene

Answer 17

B[a]P is a substrate for CYP450, which converts it to B[a]P-7,8-oxide (this is an electrophile and toxic)

The body has a defence mechanism – epoxide hydrolase converts the oxide to a dihydrodiol (B[a]P-7,8-dihydrodiol)

This is inactive

However, this dihydrodiol is also a substrate for CYP450, which converts it to another oxide (B[a]P-7,8-dihydrodiol-9,10-oxide)

This even more reactive than the previous oxide – it goes on to form DNA adducts- positively charged

The adducts can joint to chemically modified bases- and thus form covalent bonds with DNA clumping the DNA up

Question 18

Summarise aflatoxin b1

Answer 18

Formed by Aspergillus flavus mould
Common on poorly stored grains and peanuts
Aflatoxin B1 is a potent human liver carcinogen, especially in Africa and Far-East

Question 19

Describe the epoxidation of aflatoxin B1

Answer 19

§ Aflatoxin B1 epoxidation process:

o P450 oxidises the aflatoxin B1.

o Aflatoxin B1 2,3-epoxide then adducts to DNA directly using its adjacent N7 positively charged carbon atom.

Adducts on to the N7 position of the guanine-forming a mutation.

Question 20

Summarise 2-napthylamine

Answer 20

Past components of dye-stuffs
Include 2-naphthylamine and benzidine
Potent human bladder carcinogens
German dye industry epidemiology (1895 Rehn)

Question 21

Explain the mechanism by which 2-napthylamine is a bladder carcinogen

Answer 21

2-naphthylamine is converted by CYP1A2 to a hydroxylamine derivative, which is reactive (N-hydroxy-2-napthylamine)

In the liver, this is glucuronidated (thus inactivating it)- by gluconoryl transferase

The inactive metabolite is excreted by the liver and then it goes to the bladder where it mixes with the urine

The ACIDITY of the urine causes hydrolysis of the glucuronides – this releases the hydroxylamine derivative, which forms a nitrenium ion

This is electrophilic so it leads to the formation of DNA adducts

Question 22

Describe how UV radiation can cause DNA damage

Answer 22

Pyrimidine (thymine) dimers

Skin cancer

Question 23

Describe how ionising radiation can cause DNA damage

Answer 23

o Generates free radicals inside cells such as oxygen free radicals – super oxide (O2·) and hydroxyl (HO·).

o These oxygen free radicals have unpaired electrons that are electrophilic and so seek out electron-rich DNA (negatively charged sugar-phosphate backbone of DNA).

Question 24

Describe the oxygen free radical attack on DNA

Answer 24

§ Double/single stand breaks.

§ Apurinic & apyrimidinic sites – sites where the base is lost whilst the backbone remains.

§ Base modifications:

· Ring-opening – guanine & adenine.

· Glycol (unstable products of oxidation) formation – thymine & cytosine.

· Creation of 8-hydroxyadenine & 8-hydroxyguanine – mutagenic.

Question 25

Summarise the estimation rates for endogenous DNA damage and repair

Answer 25

Single-strand breaks the most common Depyrimidation least common however the max repair rate (BP/hour) per cell is greater than the rate of occurrence of endogenous DNA damage- thus allowing for genomic stability The greater the persistence of damage then the greater the chance of a mutagenic event

Question 26

Summarise how a cell responds to DNA damage and cellular stress

Answer 26

``` Detect cellular insult and then switch on genes to repair the cell. Cellular stresses can include: Oxidative stress Nitric Oxide Hypoxia Ribonucleotide depletion mitotic apparatus dysfunction oncogene activation DNA replication stress Double-strand breaks Telomere erosion ``` These insults are sensed by the tumour suppressor gene p53 P53 is normally inactivated by a partner protein called MDM2 However, these insults cause MDM2 to be lost, activating p53 and allowing it to act as a transcription factor to turn on gene expression for genes involved in DNA repair

Question 27

What are the p53 mediated responses to mild and severe physiological stress?

Answer 27

``` Mild- regulation of p53 mediated genes involved in: metabolic homeostasis antioxidant defence DNA repair growth arrest ``` Severe stress- p53 mediated protein-protein interactions which lead to apoptosis.

Question 28

Summarise the different types of DNA repair

Answer 28

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 29

Summarise DNA direct repair

Answer 29

Direct Repair involves the reversal or simple removal of the damage by the use of proteins which carry out specific enzymatic reactions Photolyases repair thymine dimers- this enzyme is activated by visible light (>300nm) absorbed by the chromophore- the enzyme is released and DNA polymerase then restores the native DNA. O6 methylguanine-DNA methyltransferase (MGMT) directly reverses some simple alkylation adducts or methyl adducts- removes these alkyl and methyl groups at their own expense and are known as 'suicide' enzymes.

Question 30

What is important about DNA mismatches

Answer 30

They are not mutations- they are simply as a result of the error rate of DNA polymerases during replication.

Question 31

Summarise DNA mismatch repair

Answer 31

Mismatch Repair involves scrutinisation of DNA for apposed bases that do not pair properly. Mismatches that arise during replication are corrected by comparing the old and new strands [proof-reading]. Preference for newly synthesized strand. Other systems deal with mismatches generated by base conversions, such as those which result from deamination.

Question 32

Outline how DNA mismatch repair takes place

Answer 32

MSH and MLH detect mismatch in DNA nucleases then excise the base and the bases downstream (preference to excise the newly synthesised strand) DNA polymerases then add the correct bases.

Question 33

Which base is most electron-rich and hence most capable of attracting electrophiles?

Answer 33

Guanine

Question 34

Outline base excision repair

Answer 34

mutagen that leads to damage to single base but no damage to backbone can be fixed by only removing base in question (can remove small adducts) DNA glycosylase hydrolyses between the base and the sugar (forming an apurinic/apyrimidinic site). Then AP endonuclease splits the DNA strand so there is a gap in the backbone DNA polymerase then fills in the missing base (using the complementary strand as template) DNA ligase then seals the DNA Re-forms the phosphodiester bonds

Question 35

outline the nucleotide excision repair pathway

Answer 35

repairing base damage by replacing nucleotides - does damage backbone (can remove larger adducts)- before they form mutations Endonuclease makes two cuts in the DNA on either side of the site of damage (this demarcates a patch of DNA) Helicase then removes this patch, leaving the double strand with a patch missing DNA polymerase replaces the missing bases DNA ligase joins the DNA up- re-forms phosphodiester bond

Question 36

State 3 human diseases involving defects in nucleotide excision repair

Answer 36

Xeroderma Pigmentosum Trichothiodystrophy Cockayne's syndrome Multiple genes involved in nucleotide excision repair- so multiple phenotypes exist.

Question 37

Describe xerdomerma pigmentosum

Answer 37

severe light sensitivity severe pigmentation irregularities early onset of skin cancer at high incidence elevated frequency of other forms of cancer (eye and G.I) frequent neurological defects Management focussed at avoiding U.V radiation, ongoing studies analysing whether shifting their activities to the night time will have any improvements.

Question 38

Summarise trichothiodystrophy

Answer 38

``` sulphur deficient brittle hair facial abnormalities short stature ichthyosis (fish-like scales on the skin) light sensitivity in some cases ```

Question 39

Summarise cockyane's syndrome

Answer 39

``` dwarfism light sensitivity in some cases facial and limb abnormalities neurological abnormalities early death due to neurodegeneratio ```

Question 40

When do double-strand breaks normally occur

Answer 40

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

Question 41

Describe homologous DNA double strand repair

Answer 41

Following a double strand break, a 3'-5' exonuclease exposes a 5' overhang (essentially a 3' tail). Transient base pairing of several nucleotides allows ends to come together DNA polymerisation (3' ends act as primers) Nucleolytic processing (hairpin formed when 3' tails displace equal strand on another DNA molecule- once it's been resynthesised- ti re-inserts between the broken strands). Ligation DNA double strand equal to the original structure is formed.

Question 42

Describe non-homologous DNA double strand repair

Answer 42

Ku proteins bind to exposed ends Recruit DNA-PKcs- which phosphorylates Artemis- ( an nuclease and phosphorylates it) which degrades the ends Ligase IV and XRC44- joins the broken ends together- forced together- even if they aren't continuous- you lose the nucleotides removed by the nucleases Therefore, the duplex that forms is not the same as the original before the break.

Question 43

Summarise the direct reversal of DNA damage

Answer 43

photolyase splits cyclobutane pyrimidine-dimers | methyltransferases & alkyltransferases remove alkyl groups from bases

Question 44

Summarise base excision repair

Answer 44

(mainly for apurinic/apyrimidinic damage) DNA glycosylases & apurinic/apyrimidinic endonucleases + other enzyme partners A repair polymerase (e.g. DNA Polβ) fills the gap and DNA ligase completes the repair.

Question 45

Summarise nucleotide excision repair

Answer 45

(mainly for bulky DNA adducts) Xeroderma pigmentosum proteins (XP proteins) assemble at the damage. A stretch of nucleotides either side of the damage are excised. Repair polymerases (e.g. DNA Polδ/β) fill the gap and DNA ligase complete the repair.

Question 46

Summarise during or post-replication repair

Answer 46

mismatch repair | recombinational repair

Question 47

Outline a schemata for the potential fates of carcinogen-DNA damage

Answer 47

Carcinogen damage leading to altered DNA: Efficient repair -normal cell Apoptosis- cell death Incorrect repair/altered base sequence -- DNA replication and cell division: fixed mutations, which can lead to: Transcription/translation- giving aberrant proteins Carcinogenesis if critical targets are mutated: oncogenes, tumour suppressor genes.

Question 48

Summarise the therapeutic agents which cause DNA damage to treat cancer

Answer 48

Agents that induce double strand breaks Ionising Radiation (Radiotherapy) Bleomycin Neocarzinostatin Agents that make bulky adducts Cisplatin Mitomycin C ``` Alkylating agents Altretamine Bendamustine Bendamustine Hcl Busulfan Carboplatin Uracil Mustard ``` Try to overwhelm cell with DNA damage such that it has to undergo apoptosis.

Question 49

outline the hierarchy for assessing the ability of a potential carcinogen/drug to cause DNA damage

Answer 49

Structural alerts/SAR In vitro BACTERIAL gene mutation assay e.g. Ames test with S. typhimurium n vitro MAMMALIAN CELL assay e.g. chromosome aberration, TK mutation in mouse lymphoma cell Micronucleus assay In vivo MAMMALIAN assay e.g. Bone marrow micronucleus test transgenic rodent mutation assay Investigative in vivo MAMMALIAN assays

Question 50

Describe the bacterial (Ames) test for the mutagenicity of chemicals

Answer 50

This test usually uses Salmonella typhimurium The bacterium is genetically engineered so that it can’t produce histidine, so it can only survive and grow on a culture medium that has exogenous histidine The compound to be tested is, firstly, incubated with rat liver enzymes containing CYP450 (S9- for phase 1 and 2 metabolism) enzymes to metabolise the chemical into an active form that can be carcinogenic The bacteria are mixed with the active chemical and then placed on a culture medium with NO histidine Any colonies that survive will have become mutated by the chemical so that it regains the ability to produce its own histidine and hence can grow in the absence of histidine Any bacteria that hasn’t been mutated will die on the dish The greater the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine the chemical may be converted to a reactive metabolite which damages the DNA and causes mutations.

Question 51

Describe the in vitro mammalian cell assay for chromosomal aberrations

Answer 51

``` Treat mammalian cells with chemical in presence of liver S9. Look for chromosomal damage: chromatid exchange chromosome gap double minutes chromosome break acentric ring chromosome interchanges ```

Question 52

Describe the in vitro mammalian cell assay for the micronucleus assay

Answer 52

This is trying to measure the ability of a chemical to break up DNA into fragments We need the cell to go through one replication cycle and then stop it when it’s at the binucleus stage – this is when you check for the presence of micronuclei Cytochalasin-B holds the cell in the binucleate stage Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss)

Question 53

Describe the murine micronucleus bone marrow assay

Answer 53

Treat animals with chemical and examine bone marrow cells or peripheral blood erythrocytes for micronuclei Bone marrow is pluripotent The animals are treated with the chemical and their bone marrow cells and peripheral erythrocytes are examined for the presence of micronuclei Erythrocytes normally remove the nucleus during development, but it CANNOT remove small fragments of DNA e.g. a micronucleus So the presence of micronuclei in erythrocytes indicates DNA damage- so look for presence of micronucelie in polychromatic cells

Question 54

What is the purpose of the tests that assess DNA damage

Answer 54

The purpose of these tests is to assess the ability of the chemical to damage DNA and thereby determine the risk to humans of the chemical causing mutation or other genomic damage. Since damage to DNA leading to mutation and dysregulation of normal genomic activity in the cell is a feature of cancer, understanding the risks to humans of exposure to chemicals is important.