DNA damage and repair Flashcards
What percentage of cancers are due to diet?
40-45%
What can damage DNA?
Chemicals: Dietary Lifestyle Environmental Occupation Medical Endogenous Radiation Ionising Solar Cosmic
Give an example of something endogenous that can damage DNA?
Mitochondria produce reactive oxygen species
What sort of DNA damage can carcinogens cause?
Base dimers and chemical cross-links
DNA adducts and alkylation
Base hydroxylations and abasic sites formed
Double and single strand breaks
What are base dimers and chemical cross links?
DNA molecules are being chemically linked up
What are base hydroxylations?
An oxidative reaction occurring on one of the DNA bases and this can cause problems as DNA might have to get repaired and during repair process, it could become mutated
What are abasic sites?
During the repair process, the entire DNA base has been removed so the sugar backbone is maintained but we have removed the base from mutagenic molecule- during replication, the missing base will cause problems
What are single strand breaks?
These are very common and useful
There are physiological enzymes that are responsible for making them
What is the role of topoisomerase?
It is involved in 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
What are double strand breaks?
These are a bit of a disaster as after the double strand breaks thee is a tendency for the two bits of DNA to drift apart and this is intolerable -there are some DNA repair mechanisms that attempt to repair this but sometimes it can go wrong and introduce DNA damage
What generally causes DNA adducts and alkylation?
Chemicals- they tend to be metabolically activated into electrophiles and DNA is very rich in electrons because of all the nitrogens in bases
What happens in DNA adducts and alkylation?
Electrophiles bind to the DNA and form a covalent bond and 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 next but can’t because of the big chemical group
What are the two phases of mammalian metabolism?
Phase I:
Introduction or unmasking of functional groups that can be used in phase 2 (mainly cytochrome p450-mediated)
Phase 2: Conjugation of phase I functional groups with endogenous molecule to make it water soluble so it can be excreted in urine
What is the whole purpose of metabolism?
Take something that is lipophilic and make it more polar so that we can get rid of it
What are polycyclic aromatic hydrocarbons?
Common environmental pollutants (lot in smoke)
What is B[a]p?
Substrate for CYP450 which oxidises it to form an oxide (Benzo[a]pyrene-7,8-oxide)
What is the B[a]p oxide like?
It is reactive and wants to find electrons
What is the body’s defence mechanism against B[a]p oxide and other polycyclic aromatic hydrocarbon oxides?
Epoxide hydroxylase cleaves the three membered strained ring of the oxide to form a dihydrodiol- not toxic
What happens to the dihydrodiol that is formed?
It is unfortunately a substrate for p450 as well so p450 converts this non-toxic metabolite into another oxide which is very reactive and desperate to find some electrons to react with and best source of electrons is DNA so DNA adducts are formed
What are 2-napthylamine and benzidine?
Part component of dye stuffs and potent bladder carcinogens
How do polycyclic aromatic hydrocarbons cause cancer in many parts of the body?
P450 is involved in its activation and found in lots of different tissues
What do aflatoxins primarily target?
The liver because it is mainly activated by p450 that is found in the liver
What is 2-naphthylamine a substrate for?
CYP450 which converts the amino group to form a hydroxylamine (N-hydroxy-2-naphthylamine)- they are reactive
What happens to the reactive hydroxylamine in the liver?
It is glucuronidated (detoxified) so chemical is activated and inactivated
What happens to the glucoronide in the urine?
Urine is acidic so glucuronide is hydrolysed and this releases the hydroxyl amine derivative which in acidic conditions rearranges to form a positively charged nitrogen (nitrenium ion)
What does the nitrenium ion do?
It is an electqrophil which then goes and binds to DNA and forms adducts- bladder isn’t as capable at detoxifying the hydroxyl amine derivative as the liver
What can UV radiation lead to the formation of?
Pyrimidine dimers- under UV radiation they can covalently link
What is the main type of cancer that UV radiation causes?
Skin cancer
What examples of ionising radiation are there?
Gamma
X-rays
Beta particles
What does ionising radiation have the ability to generate?
Chemistry within a cell- oxygen free radicals
What is a super oxide radical?
It is a molecule oxygen that has an extra electron so is very reactive
What is a hydroxyl radical?
A hydroxyl group that has grabbed an extra electron- even more reactive than super oxide radical
What are abasic (apurinic/apyrimidinc) sites?
Base has been oxidised by an oxygen free radical and DNA repair enzymes come and cut out the base itself, leaving the sugar-phosphate backbone intact so there are gaps
What base modifications are there?
Ring-opened guanine and adenine
Thymine and cytosine glycols
8-hydroxyadenine and 8-hydroxyguanine
What is the role of p53?
It is a crucial tumour suppressor gene
How does p53 normally exist?
It is tied up with MDM2 which keeps p53 inactive
What happens when p53 is released from MDM2?
It forms a dimer that activates want pathways
What happens in terms of p53 when 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
What happens in terms of p53 when we have severe physiological stress?
p53 can activate an apoptotic pathway by directly interacting with apoptosis proteins
What different types of DNA repair are there?
Direct reversal of DNA damage
Base excision repair
Nucleotide excision repair
During or post-replication repair
How many genes code for DNA repair?
Over 100
What does photolyase do?
Looks specifically for cytlobutane-pyrimidine dimers and cuts them (generated by solar radiation in first place)
What do methyltransferases and alkyltransferases do?
Remove alkyl groups from bases
What is base excision repair mainly for and what is it?
Abasic damage- DNA glycosylases and abasic endonuclease are interested in repairing this damage
Repair polymerase fills the gap caused by the missing base and DNA ligase repairs it
What is nucleotide excision repair mainly for?
Bulky DNA adducts
How does nucleotide excision repair work?
Xeroderma pigmentosum proteins assemble at site of damage- stretch of nucleotides on either side of damage are excised
What if people are defective in xeroderma pigmentosum enzymes?
They can’t repair the damage from bulky adducts and are prone to cancer
Which base is the most electron rich and what happens as a result of this?
Guanine (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 must be removed
What is the base excision repair pathway?
DNA glycosylase split/hydrolyses between the sugar and DNA base
Then an AP-endonuclease splits the DNA strand so there is a gap in the backbone
DNA polymerase then fills in the missing base
DNA ligase then seals the DNA to form intact DNA
What is nucleotide excision repair?
Endonuclease makes two cuts in DNA on either side of site of damage
These patches can be long or short
Helicase will then remove the patch, leaving the double stranded DNA with a patch missing
DNA polymerase replaces bases that have been removed using complementary strand as template
DNA ligase joins DNA up again
This process is energy demanding and requires lots of proteins
What happens if DNA damage is excessive?
Cell will commit to apoptosis
In terms of DNA damage when do most problems occur?
Between excessive and small amounts of damage- could lead to incorrect repair/altered primary sequence
How does incorrect repair or an altered primary sequence lead to carcinogenesis?
DNA replication and cell division will then mean that we have fixed damage in the daughter cell (permanent mutation), this can lead to transcriptional and translation problems leading to formation of aberrant proteins or carcinogenesis
What is the first stage in testing for DNA damage?
You look at the structure of the chemical to see if there are any functional groups that could cause problems
What is the simplest way to see whether an agent can cause mutation?
Introduce it to bacteria and see whether it causes mutation and if it causes damage to DNA of bacteria, it can damage DNA of mammals
After testing the agent on bacteria, what is the next stage of testing to see if an agent can cause mutation?
Test it on mammalian cells (in vitro)- more sophisticated genetic material (e.g. histones and chromosomes) than bacteria
What is the next stage in testing after in vitro mammalian cells?
In vivo on mammals using bone marrow micronucleus tests and transgenic rodent mutation assays (very expensive)
Why is bone marrow used for in vivo testing?
It contains pluripotent stem cells that give rise to cells of the blood so you can look at formed elements of blood as what is happening in bone marrow and what effect the chemical is having on it
What test is used for mutagenicity of chemicals?
Bacterial (Ames) test
What bacteria is normally used for Ames test for mutagenicity of chemicals?
Salmonella typhimurium
What needs to happen for the chemical in question to mutate the bacteria?
The chemical has to be metabolically activated (which usually requires P450 involvement
How do you activate the chemical in question in a lab?
You incubate it with a preparation of rat liver enzymes (containing p450 enzymes) and that generates the reactive chemical
Why are bacteria used in the Ames test genetically engineered?
They are engineered so they can’t produce histidine (amino acid) which means that these bacteria require exogenous histidine to be able to grow however, using chemical mutagens, these cells van be mutated to regain the ability to produce histidine so can grow in the absence of it
What will the results of the Ames test show?
Anything that hasn’t mutated will need exogenous histidine to grow and hence will die on the plate and bacteria that have been mutated and regained the capacity to produce histidine will grow and survive-
More the DNA damaging capability of the chemical, the more colonies will grow in absence of histidine
What is trying to be achieved in in vitro micronucleus assays?
Mammalian cells are treated with chemicals in vitro and allowed to dive- we are trying to measure ability of chemical to break up DNA into fragments, then we count the fragments
What does the cell need to do in vitro micronucleus assays?
We need the cell to go through one replication cycle and then stop it when a binucleus is formed
What is cytochalasin-B used for?
It is used to block cytokinesis and hold the cell in the binucleate stage
What are the binucleate cells assessed for?
Presence of micronuclei
What are the kinetochores of chromosomes stained to determine?
If chemical treatment caused:
Clastogenicity- chromosomal breakage
Aneuploidy- chromosomal loss/change in number of chromosomes
Both of which can contribute to cancer
How does a bone marrow micronucleus assay in mice or rats work?
Animals are treated with chemical and bone marrow cells or peripheral erythrocytes are examined for presence of micronuclei
What do erythrocytes normally do in terms of the nucleus?
They normally remove the nucleus during development but it can’t remove small fragments of DNA so if chemical can generate small fragments of DNA as erythrocytes are formed from pluripotent stem cells, these fragments will persist
