L 2: Molecular mechanisms of DNS and chromosomal damage and repair Flashcards
1
Q
Double stranded DNA structure
A
- double starnded, attached by hydogen bonds
- Bases: 2 Purines ( A/G) and 2 pyramidines (C/T)
- Base pairs: A=T, C=G
2
Q
DNA damage after a dose of 1Gy
A
- DS breaks: 40
- SS Breaks: 1,000
- Base damage >2000
- DNA-DNA Crosslinks 30
- Ratio of SS to DS DNA breaks: 25
3
Q
Diameter around DNA to cause damage
A
- 4nm
- twice the DNA diameter (2nm)
4
Q
Spurs
A
- 4nm
- 3 ion pairs
- contains upto 100eV of energy
- Mainly for x-rays and gamma rays
- SParsely ionizing - low LET
5
Q
Blobs
A
- 7nm
- 12 ion pairs
- Contains upto 100-500eV of energy
- Mainly for densely ionizing radiations like alpha
6
Q
How to measure DNA strand breaks
A
- Pulsed field gel electrophoresis (PFGE): zig-zag pattern
- Single cell gel electrophoresis (commet assay)
- SS DNA breaks: Alkaline Elution, can detect damage from even 1cGy.
- DS DNA breaks: Neutral Elution
- Alkaline elution can detect both SS and DS but neutral can detect only DS
- Micronucleus assay: chromosomal injury (dose responsive) post radiation by counting the number of micronucleus in the cell
7
Q
DNA in the cell is protected from radiation by
A
- Free radicals next to DNA
- Protection form packaging proteins like histones
8
Q
Histone proteins
A
They are rapidly phosphorylated in response to radiation damage to form
r-H2AX.
This is mapped on western blot or other tests to find the DNA damage.
9
Q
Base excision repair
BER
A
- This is the repair for base damage.
- U removed by DNA glycosylase
- UU removed by APE-1
- repair systhesis by DNA polymerase
- Excess hanging flap removed by flap endonuclease 1 FEN-1
- DNA strands are sealed by DNA ligase.
10
Q
Nucleotide excision repair
A
- Removes bulky adducts in DNA sucha a pyrimidine dimers
- 2 pathways:
1. GGR/GG-NER
2. TCR/TC-NER - The mechanism of GGR and TCR differs only in detection of the lesion.
- The reminder of the pathway is same for both.
1. Damage recognition
2. DNA incisions usually 24-32 nucleotides in length
3. Removal of damaged region
4. Repair systhesis to fill the gap
5. DNA ligation - Defective NER leads to increase in UV induced DNA damage.
- Eg: Xeroderma pigmentosa: hypersensitive to UV damage.
- Eg: Cockayne syndrome, trichothiodystrophy
11
Q
GGR/GG-NER
Global genome repair
A
- Lesions can be removed globally - DNA that encodes or DNA that does not encode for genes.
12
Q
TCR/TC-NER
Transcription-coupled repair
A
- Can remove lesions only in DNA strands of actively transcribed genes are present.
- When DNA is actively repairing anf trancribing, if there is any damage that is blocked by RNA polymerase.
- This RNA polymerase is removed by the TCR to allow repair protiens access.
13
Q
DNA DS break repair
A
Two pathways:
1. NHEJ: Non-homologous end joining
2. HRR: Homologous recombination repair
- 53BP1 relugates what is repaired by NHEJ and HRR.
- Depends on the phase of the cell cycle
14
Q
NHEJ
A
- After DNA has been damaged, activates group of sensors mainly to start repair process and prevent the DNA to enter the cell cycle with damaged DNA.
- Occurs in G-I phase of cell cycle
- Sensors first produced are ATM & ATR that belong to PIKK family.
- ATM: promotes processing broken DNA ends to generate recombinant SS DNA by NBS/MRE11/Rad50.
- **53BP1 is the inhibitor **of HRR
- Mutations in BRACA1/BRACA2 or PARPP inhibitors inhibit HRR
- Steps in NHEJ:
1. End recognition by **Ku ** binding.
2. recruitment of DNA dependent protein kinase subunit ( DNA-PKcs).
3. End processing.
4. Fill in synthesis or end binding.
5. Ligation: DNA Ligase IV is an important component of NHEJ - Eg: SCID (immunodeficiency, DNA ligase IV is deficient, protein ARTEMIS defect)
15
Q
HRR
A
- Requires undamaged DNA strand that serves as a template for repair.
- Predominant pathway in eukaryotes (yeast).
- Error free process as it has a template to copy from
- For mammals if the cell is in S or G2 phase HRR is the pathway for repair: has the undamaged sister chromatid as a template
- Holliday junctions are resolved by MMS4 and MUS81
- Inability to resolve stalled replication forks: defect in HRR
- Mre11-Rad50-Nbs1 is a Nuclease
16
Q
Crosslink Repair
A
Can be of 2 types:
1. Intra strand repair
2. Inter strand repair
- Intrastrand is in one strand of DNA
- Interstrand is in between 2 strands of DNA; It can lead to complete block and cell death if unrepaired.
- Crosslinks are removed by NER before they are repaired by HRR.
- People with **Fanconi’s Anemia ** are hypersensitive to crosslinking agents.
- People with mutations in NER or HHR pathways are sensitive to crosslinking agents.
17
Q
Mismatch Repair Pathway
A
- Removes base-base mismatches that occur during replication.
- 4 steps in repair
1. Identification of the mismatch pair by sensors.
2. MMR factors are recruited.
3. Haboring the newly synthesized strand eith removal of mismatched series.
4. re-synthesis and ligation of the excised DNA - Mutations in mismatch repair can be in MSH, MLH, PSM families.
- Mutations lead to microstellite instability.
- Eg: Hereditary nonpolyposis colon cancer (HNPCC).
18
Q
Cell killing
A
- Always refers to DSB
- DSB in DNA makes them more sensitive to radiation.
- DSBs lead to chromosomal abberations.
- Dicentric/Ring/anaphase brige are all lethal to the cell.
19
Q
Which phase of cell cycle replication happens?
A
Interphase
20
Q
Immortalization and carcinogenesis is expressed by?
A
Telomerase expression
Telomerase length = molecular clock
21
Q
Chromosomal abberations in the peripheral lymphocytes can be recognised after what dose?
A
0.25 Gy
22
Q
Types of DNA Damage
A
23
Q
Types of abberations seen in metaphase
A
Two types
1. Chromosome abberations
2. Chromatid abberations
24
Q
Chromosome Abberations
A
- Occurs when cells are irradiated early in INTERPHASE, before they are duplicated
- So mainly SS DNA breaks.
- G0-G1 phase
- Single chromatin strands
25
Q
Chromatid Abberations
A
- Happens later in the INTERPHASE after S phase
- After the chromosome has duplicated, so DS DNA breaks
- G2-M phase
- 2 chromatin strands
26
Q
Three lethal abberations
A
- Dicentric: Chromosomal abberation
- Ring: Chromosomal abberation
- Anaphasic bridge: Chromatid abberation
- All are asymmetrical abberations as part of chromosome is lost
27
Q
Dicentric
A
- Damage of the ss DNA in early INTERPHASE
- production of sticky ends, joining of 2 adjacent sticky ends forming distored chromosome with 2 centeromeres hence the name DICENTRIC
- can be identified with karyotyping
- Done in M phase so that we can see the chromosome better
- Most common
28
Q
Ring
A
- Damage of the ss DNA in early INTERPHASE
- Both ends of the broken chromosome join to form a ring
- can be identified with karyotyping
29
Q
Anaphase Bridge
A
- Breaks occur in G2 phase of the cell cycle.
- can be identified with karyotyping
30
Q
Symmetric Translocation
A
- Breaks in G1 phase chromosomes
- Can be seen with FISH hybridization or chromosome painting.
- Cannot be seen with karyotyping
- Eg: Leukemia, burkitts lymphoma
- Not lethal
31
Q
Small interstitial deletion
A
- Occurs in the edge of the domain.
- Not lethal
32
Q
Inversion
A
- A type of symmetrical translocation
- Not lethal
33
Q
Lymphocytes
A
- Used as a marker for radiation exposure
- Mainly has dicentric and ring chromosomal damage
- 0.25Gy can be detected in the lymphocytes
- Mature T lymphocytes have a life span of 1500 days.
- FISH is used to detect.
- Basically measures the frequency of translocations even after 50 years of exposure. (survivors of bomb attacks)
34
Q
DNA mutations
A
Germline = Heritable
Somatic = Cancer
35
Q
Biologic marker for radition exposure
A
- 6 months = Dicentric
- years = Translocation
