L 2: Molecular mechanisms of DNS and chromosomal damage and repair

Question 1

Double stranded DNA structure

Answer 1

• double starnded, attached by hydogen bonds
• Bases: 2 Purines ( A/G) and 2 pyramidines (C/T)
• Base pairs: A=T, C=G

Question 2

DNA damage after a dose of 1Gy

Answer 2

• DS breaks: 40
• SS Breaks: 1,000
• Base damage >2000
• DNA-DNA Crosslinks 30
• Ratio of SS to DS DNA breaks: 25

Question 3

Diameter around DNA to cause damage

Answer 3

4nm
* twice the DNA diameter (2nm)

Question 4

Spurs

Answer 4

• 4nm
• 3 ion pairs
• contains upto 100eV of energy
• Mainly for x-rays and gamma rays
• SParsely ionizing - low LET

Question 5

Blobs

Answer 5

• 7nm
• 12 ion pairs
• Contains upto 100-500eV of energy
• Mainly for densely ionizing radiations like alpha

Question 6

How to measure DNA strand breaks

Answer 6

• 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

Question 7

DNA in the cell is protected from radiation by

Answer 7

1. Free radicals next to DNA
2. Protection form packaging proteins like histones

Question 8

Histone proteins

Answer 8

• 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.

Question 9

Base excision repair
BER

Answer 9

• This is the repair for base damage for single nucleotide.
• 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.

Question 10

Nucleotide excision repair

Answer 10

• 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

Question 11

GGR/GG-NER
Global genome repair

Answer 11

• Lesions can be removed globally - DNA that encodes or DNA that does not encode for genes.

Question 12

TCR/TC-NER
Transcription-coupled repair

Answer 12

• 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.

Question 13

DNA DS break repair

Answer 13

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

Question 14

NHEJ

Answer 14

• 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 inhibitorof 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)

Question 15

HRR

Answer 15

• 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
• 53BP1 inhibits HRR

Question 16

Crosslink Repair

Answer 16

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.

Question 17

Mismatch Repair Pathway

Answer 17

• 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).

Question 18

Cell killing

Answer 18

• 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.

Question 19

Which phase of cell cycle replication happens?

Answer 19

Interphase

Question 20

Immortalization and carcinogenesis is expressed by?

Answer 20

Telomerase expression
Telomerase length = molecular clock

Question 21

Chromosomal abberations in the peripheral lymphocytes can be recognised after what dose?

Answer 21

0.25 Gy

Question 22

Types of DNA Damage

Answer 22

Question 23

Types of abberations seen in metaphase

Answer 23

Two types
1. Chromosome abberations
2. Chromatid abberations

Question 24

Chromosome Abberations

Answer 24

• Occurs when cells are irradiated early in INTERPHASE, before they are duplicated
• So mainly SS DNA breaks.
• G0-G1 phase
• Single chromatin strands

Question 25

Chromatid Abberations

Answer 25

* Happens later in the INTERPHASE after S phase * After the chromosome has duplicated, so DS DNA breaks * G2-M phase * 2 chromatin strands

Question 26

Three lethal abberations

Answer 26

1. **Dicentric**: Chromosomal abberation 2. **Ring**: Chromosomal abberation 3. **Anaphasic bridge**: Chromatid abberation * All are asymmetrical abberations as part of chromosome is lost

Question 27

Dicentric

Answer 27

* 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

Question 28

Ring

Answer 28

* Damage of the ss DNA in early INTERPHASE * Both ends of the broken chromosome join to form a ring * can be identified with karyotyping

Question 29

Anaphase Bridge

Answer 29

* Breaks occur in G2 phase of the cell cycle. * can be identified with karyotyping

Question 30

Symmetric Translocation

Answer 30

* 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

Question 31

Small interstitial deletion

Answer 31

* Occurs in the edge of the domain. * Not lethal

Question 32

Inversion

Answer 32

* A type of symmetrical translocation * Not lethal

Question 33

Lymphocytes

Answer 33

* 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)

Question 34

DNA mutations

Answer 34

Germline = Heritable Somatic = Cancer

Question 35

Biologic marker for radition exposure

Answer 35

* 6 months = Dicentric * years = Translocation

Question 36

Artemis

Answer 36

* Protein in NHEJ DNA repair pathway * IT is phosprylated by the Ku/DNA-PKcs complex. * Artemis functional deficiencies can lead to **immunodeficiency** like SCID and hypersensitivity to ionizing radiation

Question 37

Genomic microsatellite instability

Answer 37

* Associated with deficits in mismatch repair pathway. * Mutations in MSH/MLH/PMS families * Associated with hereditary cancer syndromes like Lynch syndrome

Question 38

MRE-11

Answer 38

* Component of MRN complex * MRE-11-Rad50-Nbs1 * Defects can lead to hypersensitivity to ionizing radiation. * Seen in Ataxia telangiectasia disorder

Question 39

APE-1

Answer 39

* Apurinic endonuclease 1 * Removal of sugar residue as a part of base excision repair

Question 40

TF11H

Answer 40

* Transcription factor II H * Involved in nucleotide exicion repair * Imp for repairing purimidine dimers caused by UV radiation * defects in this and XPG causes hypersensitivity to UV radiation