DNA/Ch Damage & Repair Flashcards
1
Q
What is the mass of a positron and/or electron?
A
0.511 MeV
Or
9.11 x 10-31 kg
2
Q
What is the principal interaction between photons and tissue in radiotherapy?
A
The Compton effect
3
Q
What is the dependency of the photoelectric effect on atomic no, Z, and photon energy, E?
A
PE Effect ∝ Z3/E3
4
Q
Why are photons in the PE effect range undesirable for RT?
A
Since PE depends on Z3, it would result in preferential absorption of the photons by bone.
Also, the low energy results in poor tissue penetration.
5
Q
What is the order of the minimum energy required to ionize an atom?
A
10 — 25 eV
MNEMONIC: IOnize → 10
6
Q
Why are the other members of the EM spectrum, besides XR and gamma rays, not ionizing radiations?
A
They do not have enough energy to ionize any existing element on this earth.
7
Q
Where in their tracks do charged particles deposit most of their energy?!
A
They deposit it at the end of their tracks (Bragg peak)!
8
Q
How does the probability of pair production depend on photon energy and atomic number?
A
It increases rapidly with both.
9
Q
What critical structure plays an important role is protecting DNA from radiation damage?
A
Histone proteins
10
Q
Which DNA damage is most implicated in cell death?
A
double strand breaks
11
Q
What is the function of RAD51?
A
It is a protein (recombinase) which plays a role in homologous recombination repair.
12
Q
What type of biological damage occurs from exposure to UV radiation?
A
Formation of pyrimidine dimers, pyrimidine (-4) pyrimidone photoproducts, and Thymine-Thymine dimers.
MNEMONIC: UV causes “P”ho”t”osynthesis
Also, dimerization is most associated with UV rays.
13
Q
How much temperature change does radiation cause?
A
5 Gy only causes 0.0001 C change!
14
Q
What causes T-T dimer formation?
A
UV Rays
15
Q
What is the frequency of each type of damage caused by radiation?
A
Base damage > single-strand breaks > double-strand breaks
16
Q
What is the atomic source of XRs?
A
They are produced extranuclearly.
17
Q
What is the atomic source of gamma rays?
A
They are produced intranuclearly.
18
Q
What causes damage in the case of neutron bombardment?
A
It is the recoil proton.
19
Q
What is LET?
A
Linear energy transferred
It is the amount of energy transferred per unit of length traveled.
20
Q
How does DNA damage compare between high LET and low LET radiation?
A
High LET radiation causes much more damage than low LET radiation for the same dose.
21
Q
Are most therapeutically used radiations (gamma, electrons, photons, etc) high or low LET?
A
Low LET.
22
Q
What are some examples of high LET radiation?
A
- α particles
- Carbon ions
23
Q
What is the Compton Scatter?
A
Photon interacts with an outer shell electron. The photon and the electron are scattered.
24
Q
Which electron shell does a PE effect photon interact with?
A
Inner shell!
25
How do PE effect, Compton scatter, and pair production compare to one another?
26
How does Ra-226 decay?
It decays into Radon (Rn) and an α particle
27
Why is the alpha particle considered so dangerous?
If we breathe an alpha particle, there is no real way to remove it. It can sit there and cause damage for years and years.
28
What is the direct action of radiation?
Direct interaction with and damage to the critical targets.
For high LET radiation, Most (NOT ALL) of the damage is due to direct effects.
29
What is the indirect effect of radiation?
Production of free radicals, which can diffuse and cause damage.
For **low LET radiation, 2/3 (67%)** of the damage is due to **indirect** effects. The remaining 1/3 is 2/2 direct effects.
30
What is the diameter of DNA?
2 nm
31
What is the diameter around DNA in which a water molecule (hydroxyl radicle) must be produced to inflict damage to the DNA?
4 nm
32
What feature of a radioprotector contribtues to its action?
The sulfhydryl group (-SH)
It is present in glutathione (GSH)!
33
How long before RT should patients stop taking anti-oxidants?
2-3 weeks!
34
What is the conversion between Rads and Gray?
100 Rads = 1 Gy
or
1 Rad = 1cGy
35
What are the main conversions between the different units of radiation?
36
What are the main conversions between different units of radiation?
37
What is the average natural background radiation exposure per year in the US?
3 mSv
38
What is the paradigm of radiation injury?
39
What kind of double-strand break is error-free?
Homologous recombination: This uses the duplicate of a chromosome (identical chromatid) as a template to repair the DS break. This happens during the late S and G2 phases of the cell cycle, when chromosomes are duplicated!
40
What is the most common cell cycle phase in a tumor?
G1 phase
41
What is the most common mechanism of DS DNA break repair in tumor cells?
Since most cells are in G0, and G1 phases, when identical chromatics are not available, ds breaks are repaired by non-homologous end joining. This process is very error-prone.
42
What is the most sensitive method of measuring DNA **damage** in modern times?
Comet assays
43
What is a comet assay?
It is a gel electrophoresis system for detecting DNA breaks.
44
What kind of DNA damage does Comet assay under neutral conditions pick up?
DS DNA breaks ONLY
MNEMONIC: Neutral = double-sided
45
What kind of DNA damage does Comet assay pick up under alkaline conditions?
SS DNA breaks
AND
DS DNA breaks
MNEMONIC: Single = One-sided
46
What method of measuring DNA can sense both damage and repair?
Pulse field gel electrophoresis (PFGE)
A pulsed electric current is run through a gel, causing broken DNA to migrate to one end of the gel. The extent of migration depends on the amount of damage.
47
What is a micronuclear assay?
It is a method for detecting DNA damage. It looks at cells under a microscope and measures the amount of micronuclei, which are small nuclei-like structure that contain acentric fragments.
48
What is a dicentric fragment?
It has two centromeres.
49
What is an acentric fragment?
It has lost its centromere (no centromere).
50
What is a chromosome vs. a chromatid aberration?
When damage occurs before the DNA is replicated in anticipation of cell division, it is a chromosome aberration.
If the chromosome is duplicated, usually only one of the chromatids will be damaged.
51
What is chromatid aberration?
When damage occurs after DNA replication, usually only one chromatid is damaged and it is called a chromatid aberration.
52
What lethal aberration results in the formation of dicentric chromosomes and acentric bodies?
When breaks occur in two different chromosomes and they join together inappropriately before replication. The replication process results in the formation of dicentric chromosomes (can segregate) and acentric fragments (are lost)
53
What are acentric fragments?
They lack a centromere and thus cannot attach to the microtubule apparatus.
They are lost during cell division, and they usually represent loss of a large chunk of DNA.
54
What lethal aberration results in the formation of ring chromosomes and acentric bodies?
When radiation strikes two parts of the SAME chromosome causing two breaks, the ends of the larger fragment join together, forming a ring. The smaller fragments turn into acentric bodies, which are eventually lost.
55
What lethal aberration results in the formation of an anaphase bridge and acentric bodies?
Chromatid-type aberration
Occurs in post-replication chromosomes. Breaks occur in sister chromatids, which join together. Two smaller fragments also join together, forming an acentric fragment, which is lost.
56
What lethal process results in the formation of an anaphase bridge and acentric bodies?
Occurs in post-replication chromosomes. Breaks occur in sister chromatids, which join together. Two smaller fragments also join together, forming an acentric fragment, which is lost.
57
What is a symmetrical translocation?
Exchange of material between chromosomes. Can result in oncogene activation!
58
What is chromosomal deletion?
It results in the loss of a part of the chromosome. It can result in the loss of suppressor genes and subsequent oncogenic progression.
59
What is the ratio of DS to SS breaks caused by radiation?
1:25
60
What is CDK4?
They are cyclin-dependent kinases that play a role in the progression of cell cycle from G1 to S phase.
61
What are the two proteins involved in non-homologous end joining?
Artemis and DNA-PKcs
62
What are the units/subunits of DNA-PKs?
It has a catalytic subunit and two small accessory proteins, Ku70 and Ku80.
63
What family/domain does DNA-PK belong to?
It belongs to the phosphatidyl inositol 3-kinase-like protein kinase (PIKK) family
64
What does knocking out DNA-PK in mice result in?
It causes a SCID-like syndrome, which causes radiosensitivity and immunodeficiency.
65
What are some of the characteristics of SCID mice?
- They are radiosensitive
- They exhibit low levels of NHEJ 2/2 loss of DNA-PKs
- They lack immune systems, which permits human tumor grafts to grow on/in them.
66
What kind of DNA repair can cells of patients with xeroderma pigmentosum not repair?
They cannot repair damage from UV light (pyrimidine dimers) 2/2 a lack of nucleotide excision repair.
They are NOT sensitive to ionizing radiation!
67
What's the role of MRE11 and what kind of disorder does it cause?
It is a protein (endo and exonuclease) that plays a role in DS DNA repair (both homologous and NHEJ).
A deficiency causes the ataxia-telangiectasia-like disorder.
68
What is the role of Artemis protein?
NHEJ.
It cleaves any DNA loops or hairpins that can form during NHEJ, preparing the ends for ligation by ligase.
It ARTiculates the DNA for NHEJ.
69
70
What are the first molecular signals for ds DNA breaks?
ATR
ATM
Exists as an inactive dimer in all cells
Radiation and ds DNA breaks break the dimer and activate it
71
What protein is deficient in ataxia-telangiectasia?
Ataxia-telangiectasia mutated (ATM) protein is deficient in this disease. It plays a central role in ds DNA breaks repair and regulation of the cell cycle post-radiation.
This makes these people exquisitely susceptible to damage by radiation.
Do not confuse with MRE11, which causes AT-like syndrome, NOT AT itself.
72
How are base pair damages repaired?
Base pair excision repair.
These are the most common type of damage following radiation.
73
What percentage of the population is heterozygous for mutations a/w Ataxia-telangiectasia?
1-2%
74
What is potentially lethal damage (PLD)?
- PLD is the amount of radiation damage that can be modified by the post-radiation environment
- Usually, if cell division is delayed (2/2 environment), the cells can repair this PLD. However, if they are egged on to divide, they do not repair this damage and die in the process of dividing
75
Does neutron radiation cause any sublethal damage?
Virtually none.
76
Which genetic syndrome makes people most sensitive to radiation?
Ataxia telangiectasia.
77
Which protein localizes to the site of DNA double-strand breaks, and is used as a marker of DNA damage and repair.
γH2AX, the phosphorylated histone variant of H2AX, localizes to DNA double-strand break sides rapidly after damage and acts as a docking site for other DNA repair proteins.
78
Which **proteins** first bind to the double-strand break sites during NHEJ? What's the sequence of events?
Ku70/Ku80 heterodimer
They recruit the catalytic subunit of DNA-PK
Artemis, XLF, and DNA Ligase IV a/w XRCC1 are sequentially activated.
79
What is the first signaling event at the site of a DSB that starts the DNA repair process?
Phosphorylation of H2AX at serine 139 by ATM (kinase).
80
Persistently elevated levels of γH2AX 24 hours after DNA damage signal?
Poor DNA damage repair.
81
What kind of DNA damage repair uses PARPs?
- SS DNA break repair
- PARPs are enzymes that detect single-strand breaks and initiate NER response
82
What is the minimum amount of energy required to cause an ionization event?
10-12.5 eV
83
What is D0?
It's the dose required to reduce surviving cell fraction to 37%.
84
What kind of damage does 1 Gy (or D0) inflict?
≥ 5000 base damages
1000 SS breaks
30-40 DS breaks
85
What LET is a/w clustered damage at low dose rates?
High LET
86
What is the function of X-ray complementing factor 1 (XRCC1)?
It works with DNA ligase during base excision repair. It's also involved in SS break repair. Mutations in this can make cells 1.7-fold more sensitive to radiation.
87
What is the maximum RT dose that can be determined through peripheral blood lymphocyte assay?
4 Gy
Lymphocytes undergo rapid apoptosis and disappear after such doses.
88
What is the minimum amount of RT dose that can be determined through dicentric chromosomes in the peripheral blood?
0.25 Gy or 25 cGy
89
What is the dose relationship between the number of dicentric chromosomes and radiation dose?
Linear quadratic for low LET radiation
Linear for high LET radiation
90
What is the minimum energy required to eject an electron?
12.6 eV
91
What is the minimum energy required to cause an ion pair?
25 eV
92
How many ion pairs are present in a blob and what is its diameter?
- 12 ion pairs
- 7 nm diameter
- More common w/ high LET radiation
93
How many ion pairs are present in a spur and what is its diameter?
- 3 ion pairs
- 4 nm diameter
- More common w/ low-LET radiation
94
Do high LET radiation (densely ionizing) produce more or fewer OH radicals than low LET radiation?
Less because of extensive ion and radical recombination within spurs and blobs!
95
What is the order of the life of free radicals?
10-10
96
How soon after irradiation is H2AX protein phosphorylated?
15 mins
97
Does the formation of terminal deletions follow a linear or exponential dose-response?
It follows a linear dose response since these are single-hit lesions.
98
Is RAD52 involved in NHEJ or HR?
HR, along w/ BRCA 1/2
It recruits RAD51
99
What are some features of people with LIG4 syndrome?
100
Which enzyme is deficient in a person with LIG4 syndrome?
DNA ligase IV, which is involved in NHEJ.
101
What is the main type of repair associated with UV-induced DNA damage?
Nucleotide (not base) excision repair.
NER is defective in people with XP.
102
What gene is defective in Li Fraumeni syndrome?
p53
103
Defects in HR or NHEJ confer more radiosensitivity?
NHEJ
104
What kind of lesion leads to the formation of micronuclei?
Acentric fragments
105
What is a quadriradial aberration? Which condition has a high number of such aberrations?
It is an illegitimate interchromosomal recombination, accompanied by crossing over.
They are found in abundance in people with Fanconi anemia.
106
What is mitophagy?
It is the selective removal of damaged mitochondria.
107
What is ribophagy?
Selective removal/degradation of ribosomes.
108
What is reticulophagy?
Selective removal/degradation of the endoplasmic reticulum.
109
What is pexopathy?
Selective removal/degradation of peroxisomes
110
What is lipophagy?
Selective removal/degradation of lipids.
111
Which drugs are FDA approved for the inhibition of autophagy?
Chloroquine and hydroxychloroquine (anti-malarial drugs)
112
What is a functional endpoint within the context of radiation response?
It is an endpoint that is downstream of clonogenic survival and can include:
- organ/tissue function
- toxicity
- whole animal survival
113
What is a clonogenic endpoint within the context of radiation response?
An endpoint that directly measures the replicative capacity of cells
114
Between α and β, which represents lethal damage, and which represents sublethal damage?
α = lethal, unrepairable damage indep of fx or dose rate
β = sublethal damage, depends on fx and dose rate
β may become 0 if RT is given at a low dose rate over extended periods of time, giving the cells enough time to repair sublethal damage. Alternatively, high dose rates increase β.
115
What is the spleen colony assay?
Measures radiation sensitivity of bone marrow cells in vivo.
116
Are Xeroderma Pigmentosum patient cells sensitive or resistant to RT?
They have normal sensitivity to RT, but are relatively more resistant than people with syndromes that lead to defects in DNA damage repair.
117
How does β (L-Q model) depend on dose rate?
The higher the dose rate, the larger the β
The lower the dose rate, the smaller the β (can even be 0)
118
How do the timelines of the 4 R's of radiation compare with one another?
Repair - hours
Redistribution - multiple hours
Reoxygenation - hours-days
Repopulation - days
119
What is the equation for a purely exponential survival curve?
L-Q model → S = e -αD
Target theory model → S = e -D/Do
120
What is the equation for the single target, single hit model?
SF = e -D/D0
121
What is the multi-target, single-hit model?
Each cell has multiple independent targets, all of which must be hit to kill the cell.
It applies to mammalian cells irradiated with low LET radiation.
122
What is the equation for the multi-target, single-hit model?
SF = 1 - (1-e -D/Do)n
123
What is a typical value for Do?
Do ~ 1 Gy
124
How is D10 related to Do?
D10 = 2.3 x Do
125
Conceptually, what does Dq represent?
A cell's repair capacity
126
Conceptually, what does D0 represent?
A cell's radiosensitivity
127
What is Cockayne syndrome?
Makes pts photosensitive
Does not increase cancer risk
It's COCKY but does no real harm
128
What is effective Do?
It applies to multi-fraction regimens.
D effo = - ln(SFD) / D
where D is the fraction size
D effo is always larger than Do
129
What are some features of people with LIG4 syndrome?
- Radiation sensitivity
- Immunodeficiency
130
What is microhomology-mediated end-joining (MMEJ)?
Cells deficient in HR rely on MMEJ (previously called alternate-NHEJ) to repair dsDNA break repairs.
The damaged portion is resected, and micro-sequences at the ends are identified and re-annealed. Deletions occur during MMEJ.
131
What's the sequence of events for an indirectly ionizing radiation to cause a biological effect?
- Free e- liberation from H2O (Compton process) → water ion radical formation
- Water ion radical (H2O+) combines w/ another H2O molecule → hydronium ion (H3O +) and hydroxyl (free) radical formation (OH.)
- hydroxyl radical diffuses a short distance and breaks bonds → biological 𝛥
132
What are the approximate lifetimes of a water ion radical (H2O+), hydroxyl radical (OH.), and DNA radical (R.)?
- H2O+: 10-10s
- OH.; 10-9s
- R.; 10-5s
133
How do cells decide whether ds DNA damage is repaired by NHEJ or HR?
- ATM phosphorylation and activation be MRN complex
- ATM supports the formation of γH2AX to mark sites of ds DNA damage
- 53BP1 binds to ds DNA breaks and prevents end resection → NHEJ
- BRCA**1** is upregulated in S phase, and can displace 23BP1 to promote end resection → HR
134
What is boron neutron capture therapy?
- Boron is injected into a blood vessel and preferentially collects in tumor cells.
- The patient receives radiation therapy with low-energy, thermal neutrons.
- The neutrons are captured by boron, leading to the formation of Li-7, α particle, and energy, resulting in localized tumor damage.
135
What's deaminated cytosine?
- Deamination of cytosine turns it onto uracil (U), which is found in RNA and not DNA
- If this happens in DNA, it will be recognized and repaired
136
Can methylation and acetylation be part of normal covalent DNA modifications?
Yes!
137
Which cells in the body have a genomic DNA (gDNA) and complementary (cDNA) that is different that the normal body cells?
- B and T cells
- They undergo site-specific rearrangement (**VDJ recombination using NHEJ**) in order to produce a range of antibodies and identify a range of foreign DNA
-- That's why mice that lack NHEJ are immunodeficient!
138
What is the MRN complex?
- It's the ds DNA break repair sensing apparatus, composed of 3 subunits
-- Mre11: Endo & exonuclease that resects DNA ends and initiates homologous recombination
-- Rad50: Bridges DNA ends
-- Nbs1: Interacts w/ ATM
