DNA damage and repair Flashcards
1
Q
What is the knudson hypothesis?
A
That there are multiple hits required for carcinogenesis, based on the fact that familial cancers had an earlier onset than sporadic cancers and fitting with the increase of cancer due to age suggested by carl nordling
2
Q
What did Carl Nordling determine with regards to cancer biology?
A
Demonstrated that the incidence of carcinoma increases with the 6th power of ages suggesting that this is due to requirement of multiple mutations to accumulate
3
Q
How stable is the DNA molecule?
A
It has a high rate of spontaneous mutations caused by replication errors, chemical instability or chemical attack
This instability is countered by extensive repair mechanisms and the adaption of double stranded DNA causing a low genetic error rate
4
Q
What DNA repair mechanisms reverse DNA damage?
A
Photo reactivation and Alkyltransferases
5
Q
What DNA repair mechanisms excise DNA damage?
A
Base Excision repair, nucleotide excision repair and mismatch repair
6
Q
What DNA repair mechanisms deal with strand breaks?
A
Single strand break repair and double strand break repair
7
Q
What DNA repair mechanisms allow tolerance of DNA damage?
A
Replicative bypass and translesion DNA synthesis
8
Q
What familial cancer occurs due to mutation in the mismatch DNA repair mechanism?
A
Hereditary non-polyposis colon cancer
9
Q
What familial cancer occurs due to mutation in the Base excision DNA repair mechanism?
A
MUTYH-associated polyposis
10
Q
What familial cancer occurs due to mutation in the nucleotide excision DNA repair mechanism?
A
Xeroderma pigmentosum
11
Q
What familial cancer occurs due to mutation in the double stranded DNA break DNA repair mechanism?
A
Ataxia telangiectasia
AT-like disorder
Nijmegen breakage syndrome
Breast and ovarian cancer
12
Q
What familial cancer occurs due to mutation in the homologous recombination repair DNA repair mechanism?
A
Breast and ovarian cancer
Werner syndrome
Bloom’s syndrome
RIDDLE syndrome
13
Q
What familial cancer occurs due to mutation in the crosslink DNA repair mechanism?
A
Fanconi anaemia
14
Q
What is the Ames test?
A
A test developed by Bruce Ames using salmonella typhimuriunm histidine auxotrophs which could be exposed to carcinogens resulting in an increase in the number of colonies which reverted back to being heterotrophs showing that most carcinogens are mutagens
15
Q
What is the difference between a transition and transversion mutation?
A
A transition mutation is when a purine changes to another purine (or a pyrimidine to another pyrimidine) while a transversion mutation involves the exchange of a purine for a pyrimidine
16
Q
Are all carcinogens mutagens?
A
No some act through perturbing regulatory pathways such as those involved cellular proliferation and tumour promoters are often non-genotoxic
17
Q
What are tumour promoters?
A
Substances which can not induce cancer on their own but can often act in combination with other substances to increase the risk of cancer
18
Q
What are examples of non-genotoxic carcinogens?
A
Peroxisomes proliferators including phthalates whch are used as plasticisers
These bind to the peroxisome proliferator activated receptors which are nuclear receptors involved in proliferation and oxidative stress
Causing both DNA damage and hyperproliferation
19
Q
What are the assays to detect chromosomal abnormalities and aneuploidy?
A
Karyotypring often involving chromosome painting and the micronucleus test
20
Q
What does the micro nucleus test look for?
A
Chromosomes which have lost their centromere
21
Q
What are the assays which look for DNA damage?
A
Comet assay in single gel electrophoresis
Oxidised bases in DNA (especially 8-oxoGuanine)
22
Q
What are the assays to detect the DNA damage response?
A
Yh2ax Histone H2AX phosphorylation by ATM
23
Q
What are the assays to detect mutations defined at genetic loci?
A
Drug resistance such as HGPRT (Hypoxanthine/guanine phosphoribosyl transferase) which is selected for by resistance to 6-thioguanine
24
Q
What are the WHO defined groups for classifying substances as carcinogens?
A
Group 1- Carcingenic to humans
group 2 insufficient evidence
Group 2A probably carcinogenic to humans
Group 2B possibly carcinogenic to humans
Group 3 Not classifiable as to carcinogenicity to humans
Group 4 Probably not carcinogenic to humans
25
What are some exceptions to the rule of cancers being driven by mutations?
Epigenetic driven cancers such as Rhabdoid Ca an aggressive childhood cancer of the brain, kidney and other organs when sequenced these tumours often appear genotypically normal with only one common mutation in SMARCB1 which is a subunit of SNI/SNF chromatin remodelling complex
Ependymomas which are tumours of the meninges in children where sequencing revealed no common mutations but a common Cpg Methylator phenotype focused on targets of the polycomb repressive complex 2
26
Are all mutations present in tumours able to be detected by genomic techniques?
No often many of the mutations are only present in one or a few of the tumour cells and are therefore beyond the sensitivity of the assays, however common or clonal mutations which are the key drivers of the cancer will be seen in many cancers and are much easier to detect
27
What are the consequences of the genomic instability generated in cancer?
There is a very high mutation frequency in tumours
Tumour progression is influenced by Darwinian evolution at the somatic cell level
Any cancer therapy directed against a single target molecule will be unsustainable
Slowing the rate of mutation would have a major impact on cancer
28
What is unclear about the genomic instability which arises during tumour progression?
It is not clear how or when the instability arises although it is likely to be different when comparing sporadic and familial cancers
29
How can endogenous nucleases cause DNA damage?
The human genome has many LINE (Long Interspersed element) retrotranspoaons while only a small number of these remain functional they encode an endonuclease that induces DNA double strand breaks
30
How can Replication infidelity lead to DNA damage?
The energy difference between correctly paired and incorrectly paired base pairs is very small (one H bond) and would have a 1-10% error frequency
This is reduced through DNA polymerase base selection and proofreading, accessory proteins and postreplicative mismatch repair
31
How can spontaneous alteration to DNA bases lead to DNA damage?
There can be deamination where one base is changed to another or there can be depurination/depyrimidination generating an abasic site
32
What occurs to DNA in deamination?
In the slow reactions Adenine can be converted to hypoxanthine and Guanine can be converted to xanthine
In a faster reaction Cytosine can be converted to uracil
In the fastest and most significant reaction 5methylcytosine(part of the epigenetic code) can be converted to thymine resulting in a T-G misrepair
33
How has DNA evolved to maximise stability?
The replacement of Uracil with Thymine in DNA allowing U to be recognized as foreign by DNA repair mechanisms
Double stranded DNA increases the halflife of C
34
How does Depurination/Depyrimidination affect DNA bases?
There is spontaneous breaking of the deoxyribose-base or glycosidic bond with purine loss being 20 times faster than pyrimidine
This losss generates and abasic site which cause miscoding lesions and, due to the increased instability can result in strand breakage
35
What are the reactive oxygen and nitrogen species that cause oxidative damage to DNA?
Reactive oxygen and nitrogen species include OH free radical, H2O2, HOCl, ONOO-, NO2 Free radical, CO3- free radical
Of which the most important is the hydroxyl free radical
36
How is the hydroxyl free radical generated?
Ionising radiation through the radiolysis of water, super oxide from mitochondrial O2 reduction and xenobiotic metabolism
37
What are the main sites of mitochondrial superoxide generation?
Complex 1 or NADH dehydrogenase which transfers electrons from FMNH2 to Fe-S centres and then to coenzyme q which can all leak electrons to O2
Complex 3
38
How Is superoxide formed by drug metabolism via redox cycling?
Cyp 450 reductase is a one electron oxygen reductase which reduces CYPS and some xenobiotics and can generate superoxide
39
How can hydrogen peroxide be detoxified in cells?
Catalase can degrade it to water and oxygen
| Glutathione peroxidase can degrade it to it to water and GSSG
40
How can H2O2 cause DNA damage?
It does not directly cause DNA damage but instead H2O2 is reduced to hydroxyl radicals, by transition metals, commonly Fe2+ in the fenton reaction
41
How can hypocholorous acid cause DNA damage and provide a mechanistic link between inflammation and carcinogenesis?
This chemical is formed from H2O2 and catalysed by myeloperoxidase in neutrophils
It can chlorinate cytosine to form 5chloroC a 5meC analogue causing epigenetic silencing
And silencing of promoters
5ChloroCpG is a substrate for the maintenance methyltransferase DNMT1 causing methylation of the complimentary strand and perpetuating the epigenetic mutation
42
How are the reactive nitrogen species formed?
Super oxide reacts with nitric oxide to form peroxynitrate which can then react with CO2 to from the NO2 and CO3 free radicals
43
What are the protective mechanisms cells have for RONs?
Antioxidant molecules such as glutathione and ascorbate in the cytosol and vitamin E in membranes
44
Why is d8-oxoguanine an important oxidation product in DNA?
Guanine is the easiest base in DAN to oxidise and forms 8-oxoguanine which mispairs with A, and unlike most modified bases does not block DNA polymerases resulting in G to T transversion mutations
45
How can UV damage DNA?
It can form stable DNA lesions in high abundance most commonly affecting T-T dimers it is one of the most important types of DNA damage in biological systems as intense UV earlier then 0.5 billion years ago may have precluded evolution of terrestrial life
46
How can alkylating agents damage DNA?
These are electrophilic compounds that add an alkyl group to nucleophilic centres in DNA
These include endogenous electrophiles such as S-adenosyl-methionine as a methyl donor and exogenous chemicals such as natural products, pollutants and drugs
47
What are the nucleophilic centres in DNA?
N and O heteroatoms of the bases (especially N7 of guanine, O6 of guanine and N3 of adenine)
48
How does dimethylnitrosamine function?
It is a carcinogen activated by oxidative metabolism to a monofunctional alkylating agent
49
How does Temozolomide function?
It is a monofunctional alkylating agent and anticancer agent which is used as a prodrug which undergoes spontaneous decomposition to a methylating agent, it contains high bioavailability and good tissue distribution and used for the treatment of glioblastoma and melanoma
50
What are the 6causes of DNA damage?
```
Endogenous nucleases
Replication infidelity
Spontaneous alteration to DNA bases
Oxidative damage to DNA by reactive oxygen species
UV damage to DNA
Alkylating Agents
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51
What are bifunctional alkylating agents?
These are important as antitumour agents which have two alkylating sites making them potent radiomimetics sterilize cells efficiently
52
What are examples of bifunctional alkylating agents?
Nitrogen mustard
Mitomycin C
Cisplatin, carboplatin
PR-104 (a hypoxia activated nitrogen mustard)
53
What DNA repair pathways reverse DNA damage?
Photoreactivation, alkyltransferases, ligation of DNA breaks
54
What DNA repair pathways excise DNA damage?
Base excision repair, nucleotide excision repair, mismatch repair
55
What DNA repair pathways repair strand breaks?
Single strand break repair, double strand break repair
56
What cellular mechanisms allow for tolerance of DNA damage?
Replicative bypass, translesion DNA synthesis
57
How does the photoreactivation DNA reapir pathway function?
This repair pathway uses DNA phosphorylases not found in humans but found in other organisms like e. coli
These bind specifically to DNA at pyrimidine dimers (dark reaction) then uses reverses the pyrimidine dimer in a light reaction (300-500nm)
To do this the enzyme uses two chromophores a pterin which acts as a photoantenna absorbing ebergy at 389nm whichi s transferred to the FMNH2 which can initiate the reaction with the pyrimidine dimer
58
How can O-alkylated base damage be reversed?
Performed by O6-methylguanine methyltransferase with a high preference for O6-MeG over O4-MeT and acts as a suicide enzyme
This enzyme is important as O6-MeG and the T mismatch is a substrate for misrepair as a futile repair is generated with continual degradation of the new strand but no repair of the lesion resulting in a cytotoxic rather than protective response
59
What are the differential effects of loss of MGMT and MMR on cell survival?
Loss of MGMT in tumour cells causes sensitivity to methylating agents while loss of MMR in cells causes resistance to methylating agents
60
Why is there clinical interest in MGMT?
It is the major determinant in terms of tumour resistance to temozolomide and other methylating agents
06 Benzylguanine is a suicide inhibitor of MGMT And can be used to make tumours sensitive to methylating agents however as it also sensitizes normal tissues toxic effects are aimed to be avoided through use of prodrugs activated by hypoxia
61
What is AlkB?
It is a dioxygenase with an iron II catalytic site which makes use of a 2-ketoglutarate cofactor to perform direct dealkylation DNA repair in E. coli through oxidative dealkylation of N-methylated bases including 1MeA, 3-MeC, 1-MeG, 3-MeT
In humans there are 9 homologs of this enzyme including ABH2 and ABH3
62
What is base excision repair?
A repair mechanism which removes damaged bases, recognised by DNA glycosylases which then generate an abasic site through hydrolysing the Nglycosidic bind between base and sugar
APE1 endonuclease can the nact on this iste ot cleave the phosphodiester bind and generate a single strand break
There can then be new DNA synthesis with polymerase beta using the undamaged template to insert the correct base
63
What is TDG?
A DNA glycolyase found in all cells which removes T opposite G fixing T-G misreapir resulting from deamination of MeC
64
What is OGG1?
A DNA glycolyase found in all cells which removes OxoG opposite C
65
What is MUTYH?
A DNA glycolyase found in all cells which removes A opposite oxoG
66
What is ANPG (alkyl-N-purine-DNA glycosylase)?
A DNA glycolyase found in all cells which removes N-alkylpurines such as N7-methylguanine
67
How do DNA glycosylases demonstrate overlapping specificity?
Many of the DNA glycosylases will recognize the same types of damage particularly in the cease of uracil where several enzymes can detect uracil which ac noften be misincorporated during replication
68
What is the mechanism of DNA glycosylases?
They diffuse along the DNA looking for damaged bases, when one is found the DNA is kinked to flip the modified nucleoside into a pocket
The glycosidic bond is then cut and the glysylase remains attached to the site until they are displaced by the endonuclease APE1
69
How are abasic sites repaired in mammals?
APE 1 recruits DNA polymerase beta while PARP-1 also binds to the single strand break and helps to recruit polymerase beta
Polymerase beta can then perform an AP lyase in the N-terminal domain to excise the abasic sugar phosphate, the polymerase domain will then perform repair synthesis and the XRCC1 scafold domain will recruit XRCC1 which inturn recruits DNA ligase III
70
What is the link between MUTYH and colon cancer?
There are two DNA glycosylases involved in BER of 8-oxoG with MUTYH removing A opposite O
MUTYH mutations are responsible of MUTYH-associated polyposis which has a high incidence of colon cancer as a non-functional gene allows Gto T transversions in the APC gene
71
What is nucleotide excision repair?
A DNA repair mechanism that recognizes large lesions, typically covalent modifications, which distort the structure of DNA including UV pyrimidine dimers, Bulky DNA adducts (such as DNA and DNA-Protein crosslinks)
This mechanism has many similarities to base excision repair but makes use of dual incision removing 24-32 nucleotides rather than just the one seen in base excision repair
72
What is Xeroderma pigmentosum?
A genetic condition where individuals have photosensitive skin, neurological abnormalities and 1000 times the normal incidence of skin tumours
This is due to a defective nucleotide excision repair pathway
73
What is the mechanism of mammalian nucleotide excision repair?
There is recognition of the helix distorting lesions by XPC-Rad23-centrin2 heterotrimer causing an open bubble structure involving the ATP dependent helicases XPB and XPD, RPA single stranded DNA binding and XPA scaffold protein
The dual incision nucleases attack the stable open bubble recognizing junctions between duplex and single stranded DNA
This releases the damaged DNA in an oligonucleotide
Reapir synthesis is performed by DNA polymerase gamma or epsilon
DNA ligase 1 then makes the final phosphodiester bond
74
What drug sensitivity does Nucleotide excision repair cause?
The scaffold protein XPA is the only factor whose complete loss prevents this repair mechanism with no other known effects
Low XPA activity in testicular cancer appears to make them sensitive to cisplatin
75
What is transcription coupled repair?
Transcription coupled repair is used to describe the accelerated rate of repair seen in actively transcribed regions (5-10 times higher than in nontranscribed regions)
This mechanism requires all the factors used in nucleotide excision repair except XPC as in this case recognition is achieved by arrest of RNA polymerase 2 at the lesion
76
What is mismatch repair?
A specialised excision repair mechanism to repair mispaired bases in DNA and small indels
77
What is the mechanism of mismatch repair?
The mismatch is recognized by MutS which hydrolyses an ATP to tightly bind to the mismatch, MutS then recruits MutL, an ATP replacement of the ADP allows this complex to move like sliding clamp in both directions on the DNA
They will then encounter proteins like PNCA and RFC which mark gaps like replication forks in DNA this allows EXO1 to bind to the clamp which it escorts along the DNA to excise the mismatch, DNA poymerases and ligases then close the gap
78
What does mismatch repair depend on?
Being able to identify the daughter strand in bacteria this occurs through transiently hemimethylated cytosines in CpG sequences
In mammals this seems to be through the presence of single stranded gaps with associated proteins between okasaki fragments and possibly the replication fork for the leading strand
79
What DNA damaging agents does mismatch repair confer sensitivity to?
Methylating agents such as dimethylnitrosamine, temozolomide (O6meG)
6-Thioguanine (which is methylated by S-adenosylmethionine to S6meG)
Oxidising agent
Cisplatin and carboplatin (but not oxaliplatin)
5FU and 5FUdR
80
What is the link between mismatch repair and hereditary nonpolyposis colon cancer (Lynch syndrome)?
This inherited cancer syndrome is responsible for 15% of colorectal carcinomas and is caused by 4MMR genes like MSH2, MLH1, PMS2 and MSH6
Families also show increased cancer incidence in other tissues like the ovary, stomach and endometrium
Many of the patients are individual are heterozygous and tumours have undergone loss of heterozygosity or methylation of the promoter of MMR genes
81
What is single strand break repair?
A mechanism of DNA repair which uses many of the same enzymes responsible for late stage base excision repair such as PARP1, XRCC1, polbeta, Lig3)
Sometimes the DNA ends of the break must be cleaned up to form a 3’ Oh and 5’ Phosphate through phosphorylation by DNA kinase
These lesions are readily repaired but can lead to unwelcome recombination events
82
What is PARP-1?
An abundant nuclear protein that synthesizes the polymer poly(ADP-ribose) from NAD+ as a post translational modification of acceptor proteins
The family of proteins has 18 members and there is a correlation between levels of expression and lifespan of animals
83
What is the mechanism of action of PARP-1?
The protein contains a binding zinc finger domain which can bind single stranded DNA to increase enzyme activity resulting in PARylation which is an important protein modification in DNA repair, chromatin modification, transcription or cell death pathways despite all these acceptor proteins the most important target is PARP-1 itself as it shuttles on and off the break rapidly preventing unwanted recombination events
It will recruit XRCC-1, Ligase 3 and DNA pol 3 which will repair the break
84
What drugs to PARP-1 inhibitors sensitize cells to?
Methylating agents
85
What are the main steps in non-homologous end joining?
There si recognition and stabilization of a non-homologous end-joining complexes with a Ku70/80 dimer binding within seconds followed by recruitment of other protiens including DNA-dependent protein kinases catalytic subunit (this protein has unclear role though it phosphorylates many NHEJ and autophosphorylation results in dissociation from the complex
Processing enzymes are recruited by Ku-XRCC4 scafold including many nucleases and low fidelity family X polymerases to cause DNA end processing which will continue until igation can be achieved through the low specifity ligase 4 in a complex with XRCC4
86
What are the features of Non-homologous endjoining?
The low fidelity DNA double strand break pathway is active in all cells
Ligase 4 is critical as knockouts are non-viable
Knockout of the DNA-PK components gives viable mice but prevents lymphocyte diversity generation causing immunodeficient mice
87
What are the features of double strand break repair?
These lesions are dangerously cytotoxic and mutagenic lesions
There are three repair pathways including homologous recombination, non homologous endjoining and single strand annealing
88
What is homologous recombination>?
This has a key role in generating biological diversity as well as its role in repair
This is an effective repair mechanism through its use of sequence information for an error free repair
It is important in S and G2 phase cells in mammalian cells
89
What can homologous repair use as a template for its sequence specific information?
The homologous chromosome (which may result in loss of heterozygosity) or the sister chromatid in G2 phase cells which will result in higher fidelity, this would provide a potential explanation as to why this mechanism is typically restricted to G2 phase cells
90
What is the mechanism of homologous recombination repair?
A double strand break occurs followed by invasion of a DNA molecule from either the sister or homologous chromatid this is followed by DNA synthesis, branch migration and resolution
91
What is the role of MRN in homologous recombination repair?
It is a complex of Mre11 (nuclease), Rad 50 (member of the cohesion family), NBS (primary dsb sensor which recruits the other two proteins of the complex)
This complex is the primary sensor of DNA double strand breaks and signals to ATM (Ataxia telangectasia mutated)
92
What is the role of RPA in homologous recombination repair?
Single stranded binding protein
93
What is the role of Rad52 in homologous recombination repair?
Recruited by MRN complex
94
What is the role of Rad54 in homologous recombination repair?
Helicase recruited by MRN complex
95
What is the role of Rad51 in homologous recombination repair?
Forms nucleoprotein filament that undergoes strand invasion with the assistance of BRAC2
96
What is ataxia telangiectasia?
A inherited condition where individuals are hypersensitive to radiation and have neurological abnormalities caused by ATM (Ataxia telangiectasia mutated) mutations
97
What is AT-like disorder caused by?
Mre11 mutations
98
What is Nijmegan Breakage syndrome?
NBS mutations
99
What is the function of ATM?
It exists as an inactive dimer which is activated by autophosphorylation in response to DNA double strand breaks where it can activate cell cycle checkpoints
100
What are the two mechanisms of DNA tolerance?
Gap formation and recombination
| Translesion to DNA synthesis
101
What is the gap formation and recombination method of DNA tolerance?
A gap forms opposite the lesion which is subsequently repaired by a recombination mechanism
102
What is the translesion DNA synthesis mechanism of DNA tolerance?
DNA polymerase which bypass damage
103
What is the role of Polymerase kappa in DNA tolerance?
This is highly inaccurate when replicating undamaged DNA and is error prone due to replication through lesions such as abasic sites, guanine adducts and thymine glycol
This polymerase is upregulated in many cancers
104
What does polymerase eta do which is sensible opposite DNA damage?
This is an error free bypass polymerase which specifically inserts AA opposite cyclobutane thymidine dimers
Mutations in this polymerase decrease the ability of cells to tolerate UV damage
Mutatiosn in this protein are responsible for a variant form of xeroderma pigmentosum called XP-V
105
What does Rev1 DNA polymerase do?
This encodes a template dependent dCMP transferase which inserts a C opposite an abasic site
106
What repair mechanism deals with the O6-methylguanine DNA lesion?
MGMT
107
What repair mechanism deals with the N7-methylguanine DNA lesion?
Base excision repair
108
What repair mechanism deals with the oxidised and deaminated DNA bases?
Base Excision repair
109
What repair mechanism deals with bulky DNA lesions and DNA-protein adducts?
Nucleotide excision repair
110
What repair mechanism deals with the mispaired DNA bases?
Mismatch repair
111
What repair mechanism deals with the double strand breaks?
Non-homologous end joining
112
What repair mechanism deals with the double strand breaks in S/G2 phase?
Homologous replication
