6. DNA damage and repair

Question 1

What is a carcinogen?

Answer 1

A chemical that can damage DNA and cause cancer

Question 2

What are the different factors that can increase the risk of cancer? Give examples

Answer 2

Diet - strongly associated with cancer (about 40-45% of human cancers)
Lifestyle – smoking or drinking alcohol
Environment - Herbicides and pesticides contribute to a relatively small proportion of the human cancer burden. Also, pollution
Occupational – exposure to radiation in your occupation
Medical treatments - such as radiotherapy, can also damage DNA and increase the risk of cancer. Cancer drugs also have the ability to damage the DNA – although they are used beneficially during the treatment, the drugs may cause problems in the future.
Endogenous - for example, mitochondria produce reactive oxygen species that have the ability to damage DNA
As we grow older we accumulate more and more mutations, which could lead to a neoplastic phenotype
Radiation – ionising radiation, solar radiation (especially for those with fair skin) and cosmic radiation (from space) all can cause cancer

Question 3

What are the different types of DNA damage that can be caused by carcinogens?

Answer 3

Base Dimers and Chemical Cross-Links
Base Hydroxylations
Abasic Sites
Single Strand Breaks
Double Strand Breaks
DNA Adducts and Alkylation

Question 4

What are base dimers and chemical cross-links?

Answer 4

This is where the carcinogen chemicals are literally binding onto the strands of DNA

Question 5

How can base hydroxylations cause damage to DNA?

Answer 5

An oxidative reaction occurring on one of the DNA bases and this can cause problems
This could mean that the DNA has to get repaired and during the repair process, it could become mutated (especially if the gap that is created by the removal of an inappropriate base is not filled in)

Question 6

How can abasic sites cause damage to DNA?

Answer 6

During the repair process, the entire DNA base has been removed so the sugar backbone is maintained but we have removed the base from the mutagenic molecule
During replication, the missing base will cause problems

Question 7

How can single strand breaks cause damage to DNA?

Answer 7

These are very common and can be very useful
There are physiological enzymes that are responsible for making single strand breaks
Topoisomerase is involved in the relaxing and unwinding of DNA - it works by chopping the strand of DNA and allowing the strand to unwind and we can gain access to the DNA as the strand is re-annealed
So we can deal with single strand breaks in DNA

Question 8

How can double strand breaks cause damage to DNA?

Answer 8

These are a bit of a disaster
After the double strand breaks, there is a tendency for the two bits of DNA to drift apart and this is intolerable from the cell’s point of view
There are a number of DNA repair mechanisms that attempt to amend this, but sometimes the DNA repair can go wrong and introduce DNA damage

Question 9

How can a DNA adduct cause damage to DNA?

Answer 9

This is generally the type of damage that is caused by chemicals
Some chemicals tend to be metabolically activated into electrophiles (they really wants electrons)
DNA is very rich in electrons because of all the nitrogens in the bases
The electrophiles bind to the DNA and form a covalent bond
The binding of a big bulky chemical to the DNA causes problems particularly during replication because the DNA polymerase runs along the strand and wants to figure out which base to put in next, but it won’t be able to do this if it is bound to a big chemical group
In short, DNA polymerase cannot recognise the base because of the chemical adduct

Question 10

Why is mammalian metabolism needed?

Answer 10

To take something that is lipophilic and make it more polar so that we can get rid of it

Question 11

Give an overview of the the processes that happen in metabolism

Answer 11

Phase 1: introduce or unmask functional groups that can be used in Phase 2. Phase 1 reactions are usually mediated by cytochrome P450.
Phase 2: we use the functional groups (made available by phase 1 to conjugate it with a large endogenous molecule (which is usually polar) to make it water soluble so that it can be excreted in the urine (e.g. sulphation, glucuronidation, acetylation)

Question 12

What is the function of cyrochrome P450 enzymes

Answer 12

They are a family of 57 enzymes that have a broad substrate specificity and is responsible for oxidising chemicals (very involved in Phase 1 metabolism)

Question 13

Where might polycyclic aromatic hydrocarbons be found? What does their general structure look like?

Answer 13

They are common environmental pollutants
and are produced whenever you burn fat or organic material (e.g. wood, tobacco, fossil fuels)
Their structure involves benzene rings which makes them electron rich

Question 14

What is Benzo[a]pyrene (B[a]P)

Answer 14

This is a model compound that is used to study polycyclic aromatic hydrocarbons
B[a]P is a substrate for CYP450

Question 15

Outline the two step oxidation of Benzo[a]pyrene (B[a]P)

Answer 15

B[a]P is a substrate for CYP450, which oxidises it to form an oxide(Benzo[a]pyrene-7,8-oxide)
This oxide is reactive and wants to find electrons (it is an electrophile)
There is a defence mechanism in the body - epoxide hydroxylase cleaves the three membered strained ring of the oxide to form a dihydrodiol (Benza[a]pyrene-7,8-dihydrodiol) - this is NOT TOXIC
So far, we have converted something that is potentially toxic, to something that is toxic and then detoxified it
THE PROCESS DOES NOT STOP HERE
Unfortunately, the non-toxic dihydrodiol metabolite is also a substrate for P450
So P450 converts this non-toxic metabolite into another oxide (benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide)
This is very reactive (even more so than the previous reactive oxide) and is desperate to find some electrons to react with
The best source of electrons is DNA so DNA adducts are formed. It usually binds to guanine

Question 16

What is Aflatoxin B1 and where is it commonly found?

Answer 16

Aflatoxin is formed from the Aspergillis flavus mould and is commonly found on grains and peanuts
It is a potent liver carcinogen especially in warm humid places where it is easy for it to grow such as Africa and the far East

Question 17

Outline the epoxidation of aflatoxin and show how it can act as a carcinogen

Answer 17

Aflatoxin acts as a substrate for P450 which then forms an epoxide around the carbon-carbon double bond to form a strained molecule that is reactive
When this has reacted, it forms a positively charged carbon atom which attacks guanine in DNA so it is a DNA adduct

Question 18

What are two carcinogenic compounds that used to be found in dyes?

Answer 18

2-naphthylamine and benzidine

Question 19

Why are both benzidine and 2-naphthylamine harmful for the body?

Answer 19

Both benzidine and 2-naphthylamine are potent BLADDER carcinogens

Question 20

Why do different carcinogens cause cancers in different parts of the body? Give examples

Answer 20

Polycyclic Aromatic Hydrocarbons cause cancer in many different parts of the body because P450 is involved in its activation and is found in a lot of different tissues
Aflatoxins primarily target the liver because it is mainly activated by P450 that is found in the liver

Question 21

Outline the steps involved in the metabolism of 2-naphthylamine

Answer 21

2-naphthylamine is a substrate for CYP450, which converts the amino group to form a hydroxylamine (N-hydroxy-2-naphthylamine)
Hydroxylamines are reactive
In the liver, when this reactive hydroxylamine is formed, it is glucuronidated (detoxifying reaction) so the chemical is activated and then inactivated
This glucuronidation is done by glucuronyl transferase
The inactive metabolite is excreted by the liver and it goes into the bladder and mixes with the urine
Urine is ACIDIC, and, under acidic conditions, the glucuronides are hydrolysed
This releases the hydroxylamine derivative, which, in the acidic conditions, rearranges to form a positively charged nitrogen (nitrenium ion) which can cause DNA damage

Question 22

Why does exposure to 2-napthylamine cause tumours in the bladder?

Answer 22

The nitrenium ion formed in the metabolism of 2-naphthylamine is an electrophile, which then goes and binds to the DNA in the bladder epithelium and forms adducts. This is why you are more likely to see tumours in the bladder if you are exposed to 2-napthylamine rather than tumours in the liver
The bladder isn’t as capable of detoxifying the hydroxylamine derivative as the liver

Question 23

How can UV radiation be carcinogenic? What type of cancer does UV radiation mainly cause?

Answer 23

UV radiation can lead to the formation of Pyrimidine Dimers
NOTE: Pyrimidines = cytosine + uracil + thymine (CUT)
Purines = adenine + guanine (AG)
If there are two pyrimidines next to each other, under the presence of UV radiation, they can covalently link
The main type of cancer that this causes is Skin Cancer

Question 24

Give examples of ionising radiation. How can it cause cancer?

Answer 24

Examples of ionising radiation: gamma, X-ray, beta particles
These all have the ability to generate chemistry within a cell
They can generate free radicals as they are highly penetrating
These free radicals are very electrophilic and DNA is electron-rich so they can cause DNA damage

Question 25

Give two examples of oxygen free radicals

Answer 25

Super Oxide Radical - very powerful - this is a molecule of oxygen that has an extra electron so it is very reactive
NOTE: if there is an extra electron in the outer orbit, then the molecule will be very reactive
Hydroxyl Radical - a hydroxyl group that has grabbed an extra electron - this is even more reactive than the super oxide radical

Question 26

How can oxygen free-radical attack cause damage to DNA

Answer 26

Single strand breaks - not a big deal, we can sort these out
Double strand breaks - very damaging. Takes longer to repair and is often repaired incorrectly
Apurinic and apyrimidic sites - base has been oxidised by an oxygen free radical and the DNA repair enzymes come and cut out the base itself, leaving the sugar-phosphate backbone in tact so there are gaps (abasic sites)

You can also get base modifications:
Ring-opened guanine + adenine
Thymine + cytosine glycols
8-hydroxyadenine + 8-hydroxyguanine (mutagenic as they have an extra hydroxyl group they are not recognised as the correct base and so there are errors in DNA replication)

Question 27

What is the role of p53 and how is it normally found?

Answer 27

p53 has a very important role - it is a crucial tumour suppressor gene
It is normally tied up with MDM2, which keeps p53 inactive

Question 28

What causes the release of p53 from MDM2? What happens once p53 has been released?

Answer 28

It is separated from MDM2 in response to stress stimuli – e.g. oxidative stress hypoxia, oncogene activation etc
When it is released from MDM2, it forms a dimer that activates many pathways

Question 29

What are the different actions of the p53 dimer?

Answer 29

If we have mild physiological stress e.g. DNA repair or growth arrest, p53 orchestrates a transcriptional series of events and activates proteins that help repair the problem
If there is SEVERE stress, then p53 can activate an apoptotic pathway by directly interacting with apoptosis proteins which then mediates events that take place in the mitochondria

Question 30

What are the different ways that DNA damage can be repaired?

Answer 30

Direct Reversal of DNA Damage
Base Excision Repair
Nucleotide Excision Repair
During or Post-replication Repair

Question 31

Give examples of enzymes that can directly reverse DNA damage. How do they work?

Answer 31

Photolyase looks specifically for cytlobutane-pyrimidine dimers and cuts them to restore the normal bases
NOTE: solar radiation generates these dimers in the first place
These photolyase enzymes cut out the dimers and restore the normal sequence
Methyltransferases and alkyltransferases - remove alkyl groups from DNA bases
REMEMBER: methylation and demethylation is an important way of controlling gene expression
Sometimes, you can get inappropriate methylation or alkylation and these enzymes will remove these inappropriate groups to restore the DNA structure

Question 32

How is base excision repair carried out?

Answer 32

This is for damage where the base has been removed from the DNA
DNA glycosylases (these cut the sugar part of the molecule) and apurinic/apyrimidic endonucleases (which fill in the DNA bases that have been removed) are involved in repairing this damage
Repair polymerase fill the gap caused by the missing base and DNA ligase repairs it

Question 33

When is nucleotide excision repair used and how is it carried out?

Answer 33

E.g. if polycyclic aromatic hydrocarbons form adducts, the nucleotide excision repair enzymes will recognise these bulky adducts and will try to resolve that  
Xeroderma pigmentosum (XP) proteins assemble at the site of damage and form a repair unit - a stretch of nucleotides on either side of the damage are excised 
People that are defective in these enzymes can't repair the damage from bulky adducts and are prone to developing cancer  
Repair polymerases (e.g. pol delta and beta) fill the gap left from the excision and DNA ligase completes the repair

Question 34

What are the different types of during or post-replication repair and when do they occur?

Answer 34

Mismatch repair
Recombinational repair – recombination is a normal process where chromosomes swap over genetic material when a cell divides. This may often cause mistakes
These proteins check the DNA to make sure that it is ok before the daughter cells bud off in mitosis

Question 35

Why is guanine so commonly damaged by carcinogens?

Answer 35

The most electron-rich base is guanine
NOTE: adenine is also very electron rich
If we introduce an electrophile, it will probably target guanine and form a covalent bond - this is toxic and the cell must remove this

Question 36

Outline the Base Excision Repair Pathway

Answer 36

DNA glycosylase splits/hydrolyses between the sugar and the DNA base
Then an AP-endonuclease (Apurinic/apyrimidinic endonuclease) splits the DNA strand so there is a gap in the backbone
DNA polymerase then fills in the missing base (it determines the correct base by looking at the complementary strand)
DNA Ligase then seals the DNA to form intact DNA

Question 37

Outline how Nucleotide Excision Repair is carried out

Answer 37

Endonuclease makes two cuts in the DNA on either side of the site of damage
These patches can be long (100-200 nucleotides) or short (~10-20 nucleotides)
Helicase will then remove this patch by unwinding the DNA and allowing access to the problem site. It leaves the double stranded DNA with a patch missing
DNA Polymerase then replaces the bases that have been removed using the complementary strand as a template
DNA Ligase then joins the DNA up again
This process is energy-demanding and requires a lot of proteins

Question 38

What is the easiest type of DNA damage to deal with?

Answer 38

Single-strand breaks

Question 39

How does the extent of damage to DNA determine what happens to the cell?

Answer 39

If the DNA damage is excessive, the cells will commit to apoptosis which is effective in solving the issue of damage to DNA
Small amounts of damage can be easily repaired
However, most problems occur between excessive damage and small amounts of damage
This could lead to incorrect repair/altered primary sequence
DNA replication and cell division will then mean that we have fixed damage in the daughter cells (permanent mutations)
This can lead to transcriptional and translational problems leading to the formation of aberrant proteins or carcinogenesis is critical targets are mutated (e.g. tumour suppressor genes and oncogenes)

Question 40

How do you figure out whether a chemical agent can cause DNA damage in humans?

Answer 40

There is a tiered approach to figuring out whether a chemical agent can cause DNA damage in humans
You firstly look at the structure of the chemical to see if there are any functional groups that could cause problems
The simplest way to see whether an agent can cause mutation is to introduce it to bacteria and see whether it causes a mutation
If it can damage the DNA of bacteria then it has potential to damage the DNA of mammals
Then you test it on Mammalian Cells – these are eukaryotes so have more sophisticated genetic material (e.g. with histones and chromosomes) than bacteria
It is then tested in vivo on mammals using bone marrow micronucleus tests and transgenic rodent mutation assays (these are very EXPENSIVE)

Question 41

Why is bone marrow used when testing a chemical for DNA damage in vivo?

Answer 41

Bone marrow is used in such situations because it contains pluripotent stem cells that gives rise to the cells of the blood
This means that you can look at the formed elements of the blood as a mechanism of what is happening in the bone marrow and what effect the chemical is having on the bone marrow
It is also a proliferative compartment so you are expanding the potential to cause a mutation

Question 42

What are the preparation steps that are carried out for a bacterial test of mutagenicity of chemicals?

Answer 42

Normally, Salmonella typhimurium is used for this assay
If we just take the chemical in question and add it to the bacteria, we won’t, generally speaking, get a mutation
The chemical has to be metabolically activated (which usually requires P450 involvement) - this converts the chemical into something that is damaging
You incubate the chemical with a preparation of rat liver enzymes (containing P450 enzymes) and that generates the reactive chemical

Question 43

How doe a bacterial test of mutagenicity of chemicals work to show us if a chemical is carcinogenic?

Answer 43

The bacteria are genetically engineered so that they can NOT produce histidine(amino acid)
This means that these bacteria require exogenous histidine to be able to grow
However, using the chemical mutagens, these cells can be mutated so that they regain the ability to produce histidine, thus meaning that it can grow in the absence of exogenous histidine
So you mix the bacteria with the activated chemical and put them on a plate (which doesn’t have any histidine on it)
Anything that has NOT been mutated will need exogenous histidine to grow and hence will die on the plate
The bacteria that have been mutated and regained the capacity to produce histidine will grow and survive
So the more the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine - this is a very quantitative assay

Question 44

How can Chromosomal Aberrations be used to check if a chemical damages DNA?

Answer 44

We can look at what is happening to the chromosomes themselves
Chemicals can cause double strand breaks that leads to fragmentation of chromosomes. This can be seen using a microscope and if a chemical is causing this it must be carcinogenic
This is very labour intensive investigation

Question 45

How are in vitro Micronucleus Assays carried out?

Answer 45

Mammalian cells are treated with the chemical in vitro and allowed to divide
We are trying to measure the ability of the chemical to break up DNA into fragments, then we can count the fragments
We need the cell to go through one replication cycle and then stop it when a binucleus is formed
Cytochalasin-B is used to block cytokinesis and hold the cell in the binucleate stage
Then the binucleate cells are assessed for the presence of micronuclei - bits of DNA that have been broken off and have been left behind
The kinetochores of the chromosomes can be stained to determine if chemical treatment caused:
Clastogenicity - chromosomal breakage
Aneuploidy - chromosomal loss/change in the number of chromosomes

Question 46

How is a Bone Marrow Micronucleus Assay used to test how carcinogenic a chemical is?

Answer 46

You are using the pluripotent nature of the bone marrow in producing blood cells
The animals are treated with the chemical and the bone marrow cells or peripheral erythrocytes are examined for the presence of micronuclei
The erythrocytes normally remove the nucleus during development but it CAN NOT remove small fragments of DNA (e.g. a micronucleus)
So if the chemical can generate small fragments of DNA as the erythrocytes are formed from the pluripotent stem cells, these fragments will persist

The presence of micronuclei in the erythrocytes indicates DNA damage