Maintenance of Genome Integrity Flashcards
1
Q
How does damage to DNA occur?
A
- Copying errors during DNA replication (most common cause of DNA damage)
- Spontaneous depurination
- Exposure to different agents such as
- background ionising radiation
- UV light (associated with skin cancer)
- Tobacco products (associated with lung cancer)
2
Q
What is a DNA adduct
A
A segment ofDNAbound to a cancer-causing chemical.
3
Q
What are the 5 major types of DNA repair
A
- Direct reversal of damage
- Base excision repair - corrects DNA damage caused by reactive oxygen species deamination, hydroxylation, spontaneous depurination
- Nucleotide excision repair - removes adducts that produce large distortions in DNA
- Homologous recombination repair and non-homologous end joining - repairs DNA double strand breaks
- DNA mismatch repair - repairs copy errors made during replication
4
Q
What are the consequences of unrepaired damages to DNA
A
- mutation
- Risk of cancer development
5
Q
What is the major way in which damage is done
A
- Majorly done by methylation of base pairs
- eg: Guanine
- can be methylated at 2 different sites
- 7 methyl guanine = where the guanine base is methylated on the 7th position
- This a typical sort of damage to the base.
- 7 methyl guanine causes large distortion in the DNA and causes cell death
- it causes a problem at DNA replication
6
Q
What causes methylation
A
- Methylation is caused by drugs
- e.g. alkylating drugs used to treat cancer.
- The idea of treatment with these drugs is to kill the cell by damaging the DNA.
- cause death of cell - not mutagenic
7
Q
What happens if there is alkylation on the O-6 position of guanine
A
- ETHYL METHANE SULPHONATE - a drug (mutagen)
- causes alkylation of O-6 position
- base pair change from G-C ⇒ G-T ⇒ T-A
- At the next round of replication, the Guanine on the first strand will pair with thymine again whilst the thymine on the other strand will pair with adenine
- Therefore, overall transition from: G-C ⇒ A-T
- O-6 alkyl guanine does not result in cell death but is mutagenic ⇒ CANCER
8
Q
What are the major forms of damage induced by sunshine uv light
A
- 2 forms of lesions
1. Thymine dimers (CPD)- only occurs when you’ve got 2 thymines together
- Adjacent thymidine bases become covalently linked → intracrosslink
- Causes a major distortion in the DNA and causes difficulties at replication.
- It is a mutagenic lesion
- (6-4) photoproducts
9
Q
Describe the mechanism behind UV induced DNA lesions and how they cause DNA damage
A
- Mechanism:
- In some sequences of DNA there are Adjacent thymine bases.
- In exposure to UV light they become covalently linked to form a cyclobutane ring (thymine dimer)
- The UV light results in double bonds become saturated to forms the Cyclobutane ring
- This causes a major distortion in the DNA and causes difficulties at replication.
- This is a mutagenic lesion ⇒ CANCER- Cyclobutane ring causes a distortion in the DNA so it sticks out in the DNA structure
10
Q
Summarise DNA repair, what 2 things does repair usually involve
A
- Cells have several repair systems which are usually constitutive (always switched on)
- More than 200 genes are believed to be involved in DNA repair in man.
- There are many different substrates (e.g. 7 methyl guanine, thymine dimers etc.) for repair systems.
- Broadly speaking repair involves either
- Enzymatic reversal
- Removal and replacement of damage
11
Q
Describe how UV induced dimers undergo monomerization and what this does (eg 1)
A
- UV induced dimers undergo monomerization by action of visible light and photolyase
- This is where the bonds in the cyclobutane ring are broken and restored to double bonds present in the 2 individual thymine bases.
12
Q
What are 2 other examples of enzymatic reversal, briefly explain them
A
- 06 alkyl guanine ⇒ the alkyl group is removed by Alkyl transferase
- Strand breaks in sugar-phosphate backbone can be repaired by Ligation via DNA ligase enzyme.
13
Q
What is BER ad what is it’s primary function
A
- = Base excision repair
- function is to remove damaged bases
14
Q
When does BER occur
A
- Substrates → done on single stranded dna ad occurs when:
- Spontaneous hydrolytic depurination of DNA.
- Deamination of cytosine.
- Formation of DNA adducts after exposure to reactive small metabolites.
15
Q
Describe the process of BER
A
- . Removal of base via DNA Glycosylase (breaks glycosyl bond between base and sugar)
- Removal of apurinic site (sugar + phosphate) by an apurinic endonuclease
- Addition of new nucleotides via DNA polymerase
- Ligation via a DNA ligase.
16
Q
What glycosylases are used for which DNA damage types in BER
A
- There are many glycosylases for different types of damage
- for 7-methyl-Guanine: N-methylpurine-DNA glycosylase (MPG)
- for 8-oxo-Guanine: OGG1
- for 3-methyl-Adenine: N-methylpurine-DNA glycosylase (MPG)
- for uracil incorporation into DNA: UNG1, UNG2
17
Q
Describe Nucleotide excision repair (NER)
A
- Operates on double stranded DNA:
- Cannot act on single stranded DNA e.g. does not act during DNA synthesis
- It is Non-specific:
- It recognises distortions rather than specific adducts
- unlike base excisions that recognizes specific adducts via glycosylases.
- It recognises distortions rather than specific adducts
- Will remove and repair large adducts, e.g. thymine dimers.
- Very efficient and error free
18
Q
Outline the simple process of NER
A
- Endonuclease - chops DNA on one side of dimer and then chop on other side of dimer.
- Exonuclease–removes several or tens of nucleotides.
- We now have a gap in the DNA of many nucleotides
- Polymerase – fills gap in with new nucleotides using other strand as template
- Ligase – ligates both ends.
19
Q
Desrcibe the NER process in detail
A
- UV causes damage ⇒ cyclobutane ring (this is the dimer basically, aka CPD) on DS DNA + 6-4 product
- XPC protein recognised 6-4 product but not CPD
- XPC and XPE recognise CPD
- XPA and TFIIH recruitment ‘damage verification’
- TFIIH contains XPB and XPD
- XPB and XPD - helicases - unwind DNA in opposite direction ⇒ exposing thymine dimer
- XPF and XPG recruited - nucleases - cut either side ⇒ remove dimer
- Recruitment of DNA polymerase/accessory factors ⇒ re-synthesise DNA
- DNA ligase
- different forms of XP formed by mutations of this complicated process
20
Q
What is XERODERMA PIGMENTOSUM
A
- Autosomal recessive disorder (1-4 per million)
- Patients show extreme sun sensitivity,
- skin tumours (may be hundreds), only on sun exposed parts of the body
- Neurological abnormality in some patients
- Cultured skin fibroblasts show increased sensitivity to UV light.
- Cells can be shown to have a defect in DNA nucleotide excision repair.
- so thymine dimer remains in situ
- 8 different variants - all have a specific different mutation to daughter strand gap repair/NER
21
Q
Describe what happens when we irradiate normal cells vs XP cells
A
- The normal cells would be killed by UV light to some extent
- 10% killing with 2 joules of UV light
- XP have unusual sensitivity to UV light and this causes problems in replication, causing cells to die
- killing of 60% of cells with 2 joules of UV light
22
Q
Describe repair in normal cells vs XP cells after UV exposure
A
- Graph that plots grains against UV dose
- Normal cells (top) → increase then plateaus
- Xeroderma pigmentosa cells → hardly any grains
- black spots → sites of UV damag
23
Q
So what is the defect in XP
A
- In excision deficient XP patients there is failure to excise the damage
- therefore the thymine dimer is left in the DNA
24
Q
How can mutations in XP lead to cancer
A
- XP cells show a high mutation rate.
- Mutation probably due to unexcised dimers and, therefore, incorrect bases incorporated opposite damage.
- This mutation represents a step towards cancer development.
25
What is Basal Cell Naevus Syndrome
- Occurs without a person having been exposed to UV light.
- You get lots of basal cell carcinoma.
- It caused by the inheritance of a mutated PTCH1 gene.
- in 20–30% of cases you can develop sporadic Basal Cell Carcinoma (BCC).
- The mutation that causes this is also in PTCH1 gene.
- 73% of BCCs from XP patients have mutations in *PTCH1*
26
What is daughter strand gap repair
1. Dimers remain after repair, they aren’t removed from ssDNA
2. This is a tolerance mechanism
3. Dimers are removed later from the double stranded DNA by excision repair
27
How can mutation of daughter strand gap repair lead to XP variants (XPV)
- In this case patients are not deficient in nucleotide excision repair (NER).
- They are not very sensitive to killing by UV, but cells are hypermutable by UV.
- Sensitivity to UV can be enhanced by caffeine.
- There are however defect in replication of DNA following UV exposure of cells (daughter strand gap repair).
28
Why do XP variants have defect in replication of DNA following UV exposure
- This is because patients are deficient in an enzyme called DNA polymerase eta, (hRad30).
- Specialised polymerase which can be swapped out for normal DNA polymerase and can replicate efficiently past the UV photoproduct
- This is called trans lesion synthesis.
- The patients can’t do this so instead of being able to undergo trans lesion synthesis they also have a problem that results in mutation and development of skin cancers.
- this is why XPV patients are highly mutagenic
29
What are the causes of DNA double strand breaks and what proteins are involved in repair
- Caused by errors in replication
- Also caused by Reactive oxygen species (cellular metabolism)
- Exposure to ionising radiation, X-rays, cosmic rays and some chemotherapeutic agents
- BRCA1 and BRCA2 proteins involved in DNA double strand repair.
30
When these proteins are mutated, which cancer are they associated with
associated with breast cancer.
31
Describe what BRCA1 vs BRCA2 are and their Importance
- BRCA1 and BRCA2 are both important in the cellular response to DNA damage (evidence in rats/gamma exposed cells) and are completelty different structurally
- BRCA1
- has 2 domains
- has an N terminal E3 ligase domain/ring domain
- has a BRC Terminal domain
- BRCT domains are found in many repair proteins as pairs or with an FHA domain- bind phosphorylated serine residues
- BRCA2
- there are BRC repeat motifs (about 8) which bind to Rad 51.
- These are not present in BRCA1.
- BRCA2 is also much bigger (3,418 AA vs 1,863 AA
32
What is used to measure ds breaks
- γ-H2AX (a histone) can be used to measure DS breaks following gamma radiation
- using an immunofluorescence assay
- If there is an increased sensitivity of cells to gamma rays, it means there is a defect in the DNS DS break repair
- BRCA 1/2 lacking ⇒ breaks are not solved
- (null) human cells and BRCA 1 and 2 deficient mice suggests that these proteins are involved in the repair of DNA DS break
33
How are DNA double strand breaks repaired (2 processes)
- Non -homologous end joining (joining the 2 ends together once they have broken)
- Homologous recombination repair (BRCA1/2 are involved here)
34
Outline what Homologous Recombination Repair (HRR) is and the role of BRCA1/2 here
- Catalytic activity of Rad51 is central to this form of DNA DSB repair.
- Rad51 coats SS DNA to form nucleoprotein filament that invades and pairs with homologous DNA duplex ⇒ initiating strand exchange.
- Availability and activity of Rad51 is regulated by BRCA2.
- BRCA2 shows direct interaction with Rad51 via Binding through the 8 BRC repeats in BRCA2.
- BRCA2 controls intracellular movement and function of Rad51.
35
Describe the process of HRR
1) Induction of double strand break
- detection by a protein complex called MRE11
- → binds the DNA ends and clips the ends off to reveal a little bit of single stranded DNA
- Has endonuclease and exonuclease activity so has control of dna
2) This single stranded DNA invades the the undamaged strand to find the exact position sequence position in the undamaged strand
- HRR always requires an undamaged homologous template
3) single stranded DNA binds to protein called Rad51
4) Process of invasion is mediated by the Rad51 protein and BRCA1/2.
- Rad51 is an enzyme that is able to invade the undamaged strand in the exact same region where the single stranded dna is
4) Use polymerase to synthesise DNA across where the DNA break is
5) Nucleases needed to cut the genes and then repair the gap and allow the undamaged and damaged cells to seperate
36
What triggers the release of Rad51
Release of Rad51 triggered by DNA damage by phosphorylation of Rad51 or BRCA2.
37
What is BRCA1 role in HRR
Mechanism through interaction with and removal of 53BP1 at sites of DSB, prior to resection and recombination.
38
What is the function of BRCA2 mutations
- mutations block the relocalisation of Rad51 to DNA DSBs
- absence of BRCA2 there is no ability transport Rad51 as BRCA2 is not there so you do not find the foci.
- - The image shows *BRCA2* mutations block the relocalisation of Rad51 to DNA DSBs
- In top 2 lines we have BRCA2 wild type cells and in the presence of BRCA 2 If irradiate the cells (to cause DS breaks) then the rad51 can be seen to form foci at the sites of DS breaks
- 24hrs later this reverts to what it was before irradiation – repair has occurred
39
Although both BRCA1 an BRCA2 are involved in DNA damage responses their roles are different, describe the roles of BRCA1 vs BRCA2
- BRCA2 - control of the Rad51 recombinase in homologous recombination (DNA DSB repair)
- BRCA1 occurs on a much wider front, but links upstream sensing/signalling of damage, through 53BP1 in recombination repair.
- BRCA1 has also has roles in cell cycle checkpoints.
- Cells deficient in BRCA1/2 are unusually sensitive to PARP inhibitors
40
What is PARP and what is it involved in
- poly ADP ribose polymerase
- Single stranded break repair (SSBR)
- Given to women with breast and brain cancer who have mutations in BRAC1/2
41
What can unrepaired SSBs lead to
- The formation of DSB due to collapsing replication forks
- double strand breaks → one ended double strand break = repaired by homologous recombination repair
42
What do you see if you look at chromosome damage (no. chromatid breaks) following exposure to these PARP inhibitors
- treating cells with inhibitors of SSBR will increase the number of DNA double strand breaks produced during DNA synthesis
- DNA DSBs generated during DNA synthesis are repaired with HR due to the presence of homologous template
43
PARP inhibitors in BRCA2/1 deficient cells leads to….
- increased cell damage
- decreased Rad51 foci
- BRCA1/2 null cells are more sensitive to these inhibitors.
44
Describe the survival curves for PARP inhibitors on cells
- RED = cells that lack BRCA1 and 2 = very sensitive to PARP inhibitors - line is steeper
- so then if you take these cells treated with PARP inhibitors, you can see the BRCA 1 or BRCA2 wild type cells have very little amount of damage
- In the case of BRCA 2 mutants- we've got huge amounts of damage caused by these
45
Describe how PARP1 inhibitors treat HRR deficient tumour cells
- PARP auto-ADP- ribosylation is required to remove PARP from a DNA lesion
- PARP inhibitors trap PARP on DNA, which blocks DNA replication
- Removal of Trapped PARP1/2 requires DNA end processing and homologous recombination repair of DSBs that arise as a consequence of replication fork collapse
46
What is Non homologous end joining
- second repair process that repairs DNA DSB
- there is no recombination.
47
Outline Non-Homologous End Joining
- There is recognition and cleaning up of end by DNA pK and coup proteins.
- Joining via DNA ligase enzyme.
- Essentially just rejoining process – no recombination.
- Rad51 independent
- BRCA2 is not required for DNA DSB by NHEJ
48
What normal cell process is DNA double strand break formation and resolution a part of
- This is the process that is involved in Formation of antibodies (B lymphocytes) and T cell receptor (T lymphocytes)
- V(D)J recombination - undertaken by components of NHEJ system
49
What type of process is NHEJ, explain your answer
- error prone process because it doesn’t use a template
- not an error free re-joining system
- Errors occur due to insertion of bases at random
- not good for repair but useful for making antibodies (T and B lymphocytes - V(D)J recombination
- helps increases variation of antibodies that are available
50
Describe the process of NHEJ
1) 2 strand breaks recognised by 2 proteins - Ku70/86 heterodimers - bind to the DNA ends and synapses the ends together to allow it to be repaired
2) This recruits DNA-PKcs.
3) Together with Ku- this forms the DNA-Pkcs - DNA kinase
4) Phosphorylation of different proteins and facilitates recruitment of nuclease - reveals single strand DNA
5) Microhomology based pairing - can identify similar sequences in these 2 strands and ligates them together
- problem is that if the similarity is not directly at the ends then we've got these little flaps of DNA which then get removed
6) Gap fill and ligation
51
What is DNA mismatch repair
This repairs copy errors made during DNA replication (greatest number of mutations occur due to failure to repair replication errors)
52
What is HNPCC
hereditary non-polyposis colorectal cancer
53
What does mismatch normally repair
- Repairs base-base mismatches.
- Repairs insertion deletion loops which arise as a consequence of polymerase ‘slippage’ during replication.
- Slippage causes gains or losses in repetitive DNA e.g. [C-A,C-A,C-A ] → called ‘microsatellites’ (MS).
- Unrepaired MS are called microsatellite instability (MSI) - measured using PCR test to detect a defect in mismatch repair (determine cause of colorectal cancer)
- Genes that have microsatellites in their coding region have an increased risk of mutation in HNPCC
54
What type of genes are at increased risk of mutation in HPCC
Genes that have microsatellites in their coding region have an increased risk of mutation in HNPCC - micro satellite instability
55
What is micro satellite instability
- Frameshift mutations have been observed in different genes, all affecting growth, in HNPCC cancers
- TGF-bRII, TCF4, IGF2R, BAX, MSH6, MSH3
56
What are the most frequently mutated genes in HNPCC
- Most frequently mutated genes are :*MLH1, MSH2, MSH6*
- Frameshift mutations have also been observed affecting growth in HNPCC cancers
57
What is the mutator phenotype hypothesis for HNPCC
- mismatch repair defects lead to mutation in other genes, including those known to play a role in the adenoma – carcinoma sequence.
- Therefore, the increased mutation rate is then the cause of accelerated tumorigenesis.
- The mutator phenotype plays a role in tumour progression rather than in initiation.
