DNA damage and repair Flashcards
(125 cards)
1
Q
How many damage sites does each cell acquire in its DNA per day?
A
10,000-100,000
2
Q
How may a cell reply to DNA damage?
A
Cell cycle arrest leading to replicative senescence.
Cell death.
3
Q
What arises from a mutation in a somatic cell?
A
Either cell death or cancer.
4
Q
What arises from a mutation in a germline cell?
A
Either genetic disorder or selective advantage.
5
Q
What happens when DNA damage isn’t repaired?
A
Replication/transcription are blocked.
6
Q
What happens during inaccurate repair?
A
The DNA is no longer damaged, but the base sequence isn’t what is was before, the damage has been fixed, the repair is complete but an error has been made = mutation.
7
Q
What are the 2 main consequences of DNA damage?
A
Mutation
Aging
8
Q
What is a mutation?
A
Any permanent heritable alteration in the nucleotide sequence of a DNA molecules that is passed to the progeny cells.
9
Q
How may mutations arise?
A
Errors in replication of normal DNA - DNA polymerases have high fidelity and extraordinary accuracy but they aren’t totally perfect.
Inaccurate repair of damaged DNA.
Replication of damaged DNA.
10
Q
What is the mutation rate in the human germline?
A
0.3 mutations per 10,000000000 bp per cell division.
11
Q
How many brand new mutations does a typical new born have?
A
60
12
Q
Are more mutations in a child acquired from mother or father?
A
Father - 80% of new mutations present in child are acquired from the father.
13
Q
Why is the number of new mutations in a child related to the age of a father?
A
This is related to the number of cell divisions needed to make male gametes compared to female gametes.
14
Q
How do mutations cause aging?
A
Mutations accumulate as we age, unrepaired damage is toxic and may lead to cell death or cell senescence causing loss of functional cells (inc. stem cells) and consequent biological aging.
15
Q
What is the evidence for the role of DNA damage in aging?
A
Mutations and DNA dmaage accumulate with age.
Defects in DNA repair pathways often underlie premature aging syndromes.
Cancer sufferers cured by chemotherapy with drugs that damage DNA show signs of premature aging and increased risk (3-6-fold) of a second cancer.
16
Q
What is the incidence of frailty in young cancer survivors?
A
10%
17
Q
Name two drugs that damage DNA?
A
Cyclophosphamide
Cisplatin
18
Q
What are the way in which spontaneous damage can arise?
A
- Errors in DNA replication e.g. incorporation of incorrect nucleotides.
- Deamination of cytosine.
- Depurination.
- Damage due to reactive oxygen species arising from oxidative metabolism.
19
Q
What family of enzymes protect the mitochondria from oxidative damage?
A
Dismutases.
20
Q
Are errors in replication a result of DNA damage?
A
No, they are the result of an error by DNA polymerase enzyme.
21
Q
How can errors in replication lead to mutation?
A
If the error occurs in the top strand, the bottom strand is fine and can be used as a template but when the top strand is used as a template in the next round of replication, the mismatch is used as a template leading to permanent mutation.
22
Q
What are the 2 unavoidable types of spontaneous DNA damage?
A
Deamination
Depuration
23
Q
What is deamination?
A
Deamination of cytosine is a hydrolytic reaction in which the amino group is lost from a cytosine converting the cytosine to uracil which shouldn’t be in DNA at all.
Unless repaired before replication, deamination may lead to replacement of CG pairs with TA pairs.
Deamination of cytosine generates a UG base pair, usually the U will be repaired and replaced with C again.
If not repaired, the U is read as a T so a new piece of DNA is made with a UA pair.
If that is used as a template for replication, we get a TA pair as the A is paired with a A.
24
Q
How many cytosine deaminations occur per cell per day?
A
100-200 cytosine deamination.
25
What is depurination?
Depurination is the loss of glycosidic bone between deoxyribose and either adenine or guanine.
The glycosidic bond holding adenine/guanine onto a deoxyribose is broken generating a piece of DNA chain where there is simply a base missing = apurinic site.
This is simply a spontaneous hydrolytic reaction there is no avoiding it.
26
How many depurinationns occur per cell per day?
10,000
27
What are the consequences of depurination?
There are 2 mechanisms through which depurination can give rise to mutation.
Translesion DNA polymerases are a last resort set of polymerases that can read through a damaged site and introduce a random (possibly incorrect) base at the untemplated region to repair the site.
The second way is simply to miss out the untemplated region introducing a kink into the DNA, a nucleotide has been missed out therefore this generates a small deletion.
28
How do reactive oxygen species arise?
Through oxidative metabolism.
29
What are reactive oxygen species?
They contain an oxygen with an unpaired electron making them hugely reactive and damaging.
30
What is the consequence of reactive oxygen species?
They cause >70 different types of DNA damage, one of the most common types is oxidation of guanine.
1. Formation of 8 oxo-guanine, this can still pair with cytosine but can also pair with adenine, therefore it is sometimes read as a T leading to GC bps being converted to AT.
2. Strand breaks - single strand breaks much more common than double-strand breaks.
3. Interstrand crosslinks - complementary strands become joined by covalent bonds preventing DNA polymerase/transcription machinery from being able to unwind DNA.
4. Formation of cyclopurines - a purine becomes covalently bound to carbon 5 of deoxyribose, this blocks trasncription and replication.
31
How many molecules of 8 oxo-guanine form per cell per day?
2000-3000.
32
How many single strand breaks occur per cell per day?
>10,000
33
How many double-strand breaks occur per cell per day?
10
34
How many interstrand cross-links form per cell per da?
10
35
How many cyclopurines form per cell per day?
Several hundred.
36
What are cyclopurines?
They occur when a purine becomes covalently bound to carbon 5 of deoxyribose.
37
What is a interstrand crosslink?
Joining of complementary strands by covalent bonding, a distastrous lesion that prevents unwinding of DNA by DNA replication/polymerase/transcriptional machinery.
38
What are the 3 most common mutagens we are exposed to?
1. Ionising radiation e.g. X-rays/gamma rays
2. Various chemicals e.g. polycyclic aromatic hydrocarbons.
3. Ultraviolet radiation
39
Can you get inherited mutations generated by UV?
No - UV only affects skin cells because it only affects cells directly exposed to UV light.
40
How does ionising radiation damage DNA?
It can damage DNA directly by ionising bases/damaging the sugar phosphate backbone or indirectly by reacting with water and generating reactive oxygen species e.g. hydroxyl radicals.
41
What kind of damaged ends do clusters of reactive oxygen species generate?
Dirty ends - difficult to repair due to lacking 3'OH or lacking 5' phosphate etc or base oxidation.
42
Give some examples of ionising radiation?
Particle radiation, for example from radioactive substances in the lab giving off beta-radiation.
Short-wavelength electromagnetic radiation, e.g. gamma rays, X-rays etc.
43
How many ionising particles pass through us each hour?
300 million.
44
How many cancer cases per year are thought to be the result of medical X-rays?
700
45
How do polycyclic aromatic hydrocarbons damage DNA?
They react directly with bases and commonly become covalently bonded to guanine, this alters base-pairing properties and alters the structure of DNA hence blocking transcription/replication.
46
What is the most common polycyclic aromatic hydrocarbon?
Benzylpyrene.
47
What are the 3 ways that UV induces DNA damage?
1. The formation of cyclobutane pyrimidine dimers.
2. The formation of 6-4 photoproducts between pyrimidine (about 25% the frequency of CPDs).
3. Formation of reactive oxygen species.
48
Are CPDs or 6-4 photoproducts more commonly induced?
CPDs, 6-4 photoproducts form at 25% the frequency of cyclobutane pyrimidine dimers.
49
What is a cyclobutane pyrimidine dimer and how do they cause damage?
2 covalent bonds between Cs and Ts resulting in the formation of a 4-membered ring between 2 adjacent pyrimidine residues.
These two covalent bonds distort the DNA molecules and disrupt the hydrogen bonding and base-pairing.
50
What is a 6-4 photoproduct and how do they cause damage?
A single covalent bond between the 6th atom of 1 pyrimidine ring and the 4th atom of the pyrimidine ring next door.
This distorts the DNA strcture and alters teh hydrogen bonding/base-pairing of a DNA molecule.
51
What is the result of UVA radiation?
The formation of singlet oxygen, this has 1 electron elevated to a higher energy level and has the ability to form oxo-guanine but not double-strand breaks.
52
Which type of UV is most responsible for the formation of reactive oxygen species?
UVA.
53
What is the difference between a cyclobutane pyrimidine dimer and a 6-4 photoproduct?
A cyclobutane pyrimidine dimer is 2 covalent bonds between Cs and Ts whereas a 6-4 photoproduct is a single covalent bond between the 6th atom of one pyrimidine ring and the 4th atom of another pyrimidine ring.
54
What is the order of frequency of base-pairs that see the most CPDs?
T=T > T=C > C=T > C=C
55
By what degree do CPDs/6-4 photoproducts bend DNA at the site of damage?
They introduce a small kink causing the DNA to bend 30 degrees at the site of CPD/6-4 photoproduct.
56
Why are polymerases stalled at the site of CPD/6-4 photoproduct?
DNA/RNA polymerases are adapted to have 1 base in their active site at once and a distorted base doesn't fit.
57
Define adduct?
A chemical linked to DNA via a covalent bond.
58
Which type of UV is most damaging?
UVC
59
Which type of UV never reaches the Earth's surface?
UVC - it is blocked by the ozone layer.
60
What is the wavelength of UVC?
100-290nm
61
What is the wavelength of UVB?
290-320nm
62
What % of the suns radiation that reaches the Earth is UVB?
0.3%
63
What % of the suns radiation that reaches the Earth is UVA?
5%
64
What is the wavelength of UVA?
320-400nm
65
Why is UVC the most damaging UV?
It has the shortest wavelength making it the most energetic UV and hence the most damaging.
66
How many more CPDs does UVC induce than UVB?
100x
67
How many more CPDs does UVB induce than UVA?
1000x
68
How much more potent is UVC than UVA?
100,000-fold.
69
Which UV penetrates deepest?
UVA
70
How many CPDs or 6-4 photoproducts form per second in each epidermal cell exposed to sunlight?
50-100
71
Consider a pyrimidine chosen at random, what is the probability that there is an adjacent pyrimidine such that the C could potentially form a cyclobutane pyrimidine dimer or 6-4 photoproduct?
75%
| Pu-c-Pu, Py-c-Pu, Pu-c-Py, Py-c-Py
72
Name some DNA damage repair mechanisms?
```
Base excision repair
Nucleotide excision repair
Mismatch repair
Homology directed repair
Translesion synthesis
Non-homologous end joining
```
73
Name some DNA damage repair mechanisms that are accurate returning the DNA to its original state?
Base excision repair
Nucleotide excision repair
Mismatch repair
Homology directed repair
74
Name some DNA damage repair mechanisms that are error prone?
Translesion synthesis
| Non-homologous end joining
75
Mismatched bases are repaired by what mechanism?
Mismatch repair
76
How do mismatched bases arise?
Incorporation of incorrect bases during replication.
77
How do we get uracil in DNA?
Deamination of cytosine
78
How is the presence of uracil in DNA repaired?
Base excision repair
79
How do missing bases arise in DNA?
Depurination
80
How are missing bases in DNA repaired?
Base excision repair or translesion repair
81
How is 8-oxo guanine formed?
Reactive oxygen species.
82
How are damaged bases like 8 oxo-guanine repaired?
Base excision repair.
83
How are double strand DNA breaks induced?
Ionising radiation or reactive oxygen species
84
How are double strand DNA breaks repaired?
Homology directed repair or non-homologous end joining.
85
How are structural distortions like CPDs, 6-4 photoproducts and adducts formed?
Reactive oxygen species, polycyclic aromatic hydrocarbons and UV light.
86
How are structural disortions likes CPDs, 6-4 photoproducts and adducts repaired?
Nucleotide excision repair and translesion repair.
87
How are interstrand crosslinks formed?
Some cancer drugs and reactive oxygen species.
88
How are interstrand crosslinks repaired?
ICL repair
89
What is base excision repair used to repair?
Damaged or abnormal bases .ike U arising from deamination of cytosie or 8 oxoguanine generated by Ros or missing bases generated by depurination.
*Not used to repair significant structural distortions.
90
What is the mechanism of base excision repair?
1. Damage specific glycosylase enzymes slide along DNA and recognise and excise damaged bases generated an abasic site.
2. An AP endonuclease cleaves the phosphodiester bond 5' to the abasic site making a nick in the DNA molecule.
3. DNA polymerase beta breaks the sugar phosphate backbone on the other side of the AP site and the deoxyribose and phosphate that were linked to the damaged based are lost.
4. DNA polymerase beta replaces the missing nucleotide and a DNA ligase seals the gap and repairs the bond.
91
How many different types of glycosylase enzyme are there?
At least 11 all recogising a different distortion to the base sequence.
92
What is the role of glycosylase enzymes?
Break the bone between the base and sugar-phosphate backbone to form an abasic site.
93
What kind of enzyme is DNA polymerase beta?
A lyase (also has nuclease activity)
94
What is a lyase enzyme?
An enzyme capable of breaking a bone without oxidation or hydrolysis.
95
What is important about the activity of the AP endonuclease in BER?
It leaves a 5' phosphate and a 3' OH.
| The 3'OH is used as a primer for DNA polymerase beta.
96
What kind of damage is repaired by nucleotide excision repair?
Virtually any damage that causes structural distortion.
97
Give some examples of structural distortions repaired by NER?
Cyclobutane pyrimidine dimers and 6-4 photoproducts generated by UV, adducts caused by PAHs, cyclopurines generated by Ros.
98
What is a cyclopurine?
A purine base covalently bonded to C5 of deoxyribose in the sugar-phosphate backbone that arise due to the action of reactive oxygen species or endogenous aldehydes.
99
What is the mechanism of nucleotide excision repair?
The damaged DNA/structural disortion is recognised.
The DNA is locally unwound at the site of damage specifically in the 3' direction around the site of damage by the general helicase activity of TFIIH.
2 endonucleases make a cut either side of the area that has been locally unwound (20-30nuc in length).
The damaged site is cleaved by XPF 5' of the damaged site and XPG 3' of the damaged side, this occurs about 20 nucleotides upstream and 5 nucleotides downstream releasing a fragment 22-30 nucleotides in length including the damaged site.
This generates an even bigger gap which is an ideal substrate for DNA synthesis, a DNA polymerase comes along, latches onto the 3'OH at the 3'end of the ssDNA and fills in the gap and DNA ligase seals the nicks repairing the missing phosphodiester bond.
100
Is DNA on the template strand or non-coding strand repaired more effectively?
Template strand.
101
What are the 2 types of NER?
Global genome nucleotide excision repair.
| Transcription-coupled nucleotide excision repair.
102
Describe the mechanism of global genome nucleotide excision repair?
Any structural distortion is recognsied and bound by the protein XPC which searches for regions of ssDNA on the undamaged strand.
CPDs are less distorting than most other forms of damage so XPC requires an accessory protein UV-DDB.
After this point this is the same as TC-NER...
Unwinding of DNA close to the damaged site occurs by helicase activity of TFIIH.
The damaged site is cleaved on both sides by XPF 5' of the damagedd site and XPG 3' of the damaged site, this occurs about 20 nucleotides upstream and 5 nucleotides downstream.
The 22-30 nucleotide fragment is released including the damaged site.
DNA polymerase latches onto the 3'OH at the 3' end of the ssDNA and fills in the gap.
The missing phosphodiester bond is synthesised by DNA ligase.
103
How does XPC recognise structural distortions?
It recognises and binds regions of ssDNA on the undamaged strand opposite a structural distortion.
It has a beta-loop sticking out looking for single strands with bases that aren't base-paired indicative of a structural distortion.
104
What type of damage requires an accessory protein to be recognised by XPC?
CPDs.
105
Why does XPC require an accessory protein to recognise CPDs?
CPDs are less distorting than other forms of structural damage.
106
Describe the mecahnism of transcription-coupled nucleotide excision repair?
Upon reaching a lesion/area of structural damage, RNA polymerase stalls and the stalled RNAP recruits CSA and CSB which cause RNAP to backtrack exposing the damaged site. The RNAP stalled with CSA and CSB bound acts as a signal to the cell to recruit NER machinery.
After this point this is the same as GG-NER...
Unwinding of DNA close to the damaged site occurs by helicase activity of TFIIH.
The damaged site is cleaved on both sides by XPF 5' of the damagedd site and XPG 3' of the damaged site, this occurs about 20 nucleotides upstream and 5 nucleotides downstream.
The 22-30 nucleotide fragment is released including the damaged site.
DNA polymerase latches onto the 3'OH at the 3' end of the ssDNA and fills in the gap.
The missing phosphodiester bond is synthesised by DNA ligase.
107
How do GG-NER/TC-NER differ?
In the way the DNA damage is recognised but not in mechanism.
108
In what phases of the cell cycle can homology directed repair be used?
G2 or S
109
Why can't homology directed repair always be used?
It can only be used in the S or G2 phases of the cell cycle where there is a homologous chromosome/sister chromatid available to repair.
110
What is translesion repair?
Translesion repair is used to allow replication to continue at the expense of possible mutation.
In this case, translesion enzymes pass through the damaged site without actually reading it and put in a base at random.
111
Describe the mechanism of non-homologous end joining to repair damaged DNA?
NHEJ recognises double-strand DNA breaks quickly.
A protein called Ku is recruited to the broken ends within 5 seconds of the break.
*Ku is extremely abundant and has a very high affinity for DNA and hence is recruited so quickly.
Ku then recruits DNA-protein kinase.
There is then a Ku:DNA-protein kinase complex on both damaged ends which interact to keep the broken ends close together preventing strand separation.
A battery of nucleases/DNA polymerases trim away the damaged nucleotides and DNA ligase joins the ends.
Therefore, NHEJ works at the expense of a deletion.
112
What is the biggest protein kinase in the proteome?
DNA-protein kinase
113
What % of UV induced mutations are C-T transitions?
60%
114
What is a mutation signature?
The thing that is most characteristic of a particular mutagen.
115
What is the mutation signature for polycyclic aromatic hydrocarbons?
guanine to thymine transversions.
116
Given that UV induces dimers more efficiently than between cytosines, why do UV-induced mutations alter cytosine?
Cytosines with a pyrimidine dimer undergo deamination to uracil 10^6 times more effectively than normal cytosines.
A cytosine with a dimer has a half-life of 5 days, a cytosine without a dimer has a half-life of 20,000 years.
A cpd that involves a cytosine deaminates to U and that pairs with A and is the U is then replaced with a T in the next round of replication = C to T transitions.
117
What is the name of the translesion enzyme that can accurately read through pyrimidine dimers?
DNA polymerase eta, this has a big active site so can fit the whole dimer in its active site and repairs the dimer precisely despite being a translesion enzyme.
118
Name two inherited disorders that arise from mutations affecting DNA repair mechanisms?
1. Xeroderma pigmentosum
| 2. Cockayne syndrome
119
Describe Xeroderma pigmentosum?
A rare condition caused by recessive mutations in genes involves in GG-NER lie XPC or UV-DDB compromising the ability to repair bulky damage, particularly CPDs.
Symptoms include heightened sensitivity of skin and eyes to sunlight (increased photosensitivity), 1000x increase in risk of developing skin cancers, neurological degeneration in 20-30% of patients.
Affects 1 in 400000 but good prognosis because treatment is to avoid sunlight, wear hyperprotective suncream and wear protective clothing, if this is adhered to, life expectancy can be normal.
120
What are the symptoms of xeroderma pigmentosum?
Increased photosensitivity
1000x risk of skin cancer
Neurological degeneration in 20-30% of patients.
121
Describe cockayne syndrome?
A rare disease arising from recessive mutations in genes involved in TC-NER such as CSA and CSB.
Symptoms include extreme sensitivity of skin to sunlight but no increased incidence of cancer.
Progressive neurological degeneration.
Premature aging due to death of non-proliferatingn cells.
Life expectancy approx 12 years.
Without functional CSA or CSB, RNAP remains stalled at a damaged site, it fails to backtrack so the damaged site cannot eve be accessed by GG-NER.
122
What are the symptoms of cockayne syndrome?
Extreme sensitivity to sunlight
Progressive neurological degeneration
Premature aging due to death of non-proliferating cells.
Life expectancy approx 12 years.
123
What is the life expectancy for a sufferer of cockayne syndrome?
12 years of age.
124
What is the life expectancy for a sufferer of xerodema pigmentosum?
Normal if treatment/management adhered to.
125
What are the hallmarks of aging?
1. Altered intercellular communication.
2. Genomic instability.
3. Telomere attrition.
4. Epigenetic alterations.
5. Loss of proteostasis.
6. Deregulated nutrient-sensing.
7. Mitochondrial dysfunction.
8. Cellular senescence
9. Stem cell exhausation
