Base, SSD repair는 워낙 빈번해서 임상적 의미는 없음.
DSB가 중요함. Fig2.5에서 DNA repair 확인할 수 있는 방법들을 알아두자 (gapi?) 

End joining 중 non-homologous end joining은 꼭 잘 알아두도록.
최근에는 mismatch repair가 각광을 받고 있다 (immuno-oncology에서도)
Rectal ca.의 5% 정도가 mismatch repair에 문제가 되기도. 이런 경우에는 immuno therapy만 적용해도 거의 CR이 오더라.

Chromosome의 aberration 정도를 보고 in vivo dosimetric 할 수도. 시간이 지나서 확인은 어렵겠지만.. 비파괴 검사 때 종사자들의 exposure 를 확인할 때 사용 됨.

Linear/quadratic model
Dose가 높을수록 DSB가 많을 것임을 시사 함. 

General Overview of DNA Strand Breaks

• DNA
  • Principal target for the biologic effect of radiation
  • Double helical structure, hydrogen bond
  • Single ring group: thymine and cytosine / double ring group: adenine and guanine
  • Complementary base pairs: adenine – thymine, guanine – cytosine
  • Fig 2.1 and 2.2A
• D0: a dose of radiation that induces an average of on lethal event per cell leaves 37% still viable
  • For mammanlian cell, x-ray D0: 1-2 Gy
  • No. of DNA lesions per cell after D0
    Base damage: > 1000
    Single-strand breaks (SSBs): about 1000
    Double-strand breaks (DSBs): about 40
• Single-strand breaks
  • Little biologic consequence
  • Repaired readily using the opposite strand as a template (Fig 2.2 B-C)
  • Incorrect repair (misrepair) → mutation
• Double-strand breaks
  • Breaks in the two strand are opposite or separated by only a few base pairs (Fig 2.2D)
  • The most important lesion produced by radiation
  • Result in cell killing, carcinogenesis, or mutation
  • Induced linearly with dose
• Energy deposition events : spur/blobs/short track
  • Spur: 
    Up to 100 eV of energy, involves 3 ion pairs
    4nm in diameter (about twice the diameter of the DNA double helix)
    95% of the energy deposition events of X-ray or γ-rays
  • Blobs:
    100-500 eV, 12 ion pairs, 7nm in diameter
    Much less frequent for X-ray or γ-rays
  • Locally multiply damaged site or clustered lesion: spread out up to 20 base pairs
  • In the case of densely ionizing radiation (neutron or α-particle):
    Greater portion of blobs produced, much difficult for the cell to repair

Measuring DNA Strand Breaks

• Pulsed-field gel electrophoresis (PFGE) (Fig 2.4 A)
  • Most widely used method to detect the induction and repair of DNA DSBs
  • Separation of DNA fragments according to size in the megabase-pair range
  • The fraction of DNA is directly proportional to dose
• Single-cell gel electrophoresis (commet assay) (Fig 2.4B)
  • Advantage of detecting differences in DNA damage and repair at the single-cell level
  • The amount of DNA damage is directly proportional to the migration of DNA
  • High sensitivity and specificity for SSBs and alkaline sensitive sites, and to a lesser degree DSBs
• DNA damage-induced nuclear foci (radiation-induced foci assay)
  • Complexes of signaling and repair proteins that localize to DNA strand breaks
  • Ease of protocol & both tissue sections and individual cell preparations
  • γH2AX and 53BP1 (Fig 2.5): Detected by staining, western blot, flow cytometry
    γH2AX: H2AX (histone protein) → phosphorylated to form γH2AX
    53BP1: phosphorylated at the site of DNA DSBs
  • Other proteins: ATM, RPA, RAD51, BRCA1
  • γH2AX or 53BP1 foci directly correlate with DSBs à Measured over time: reflects the kinetics of repair
  • BRCA1 & RAD1 in breast cancer
• DNA in cell is much more resistant to radiation induced damage than free DNA
  • Low molecular weight scavengers
  • Physical protection by packing with proteins

DNA Repair Pathways

• Base damage, SSBS, DSBs, sugar damage, DNA-DNA crosslinks

Base excision repair

Nucleotide excision repair

Removes bulky adducts in the DNA

DNA double-strand break repair 

Nonhomologous end-joining (NHEJ)

• Error prone & important role in generating antibody diversity
• Primarily found in the G1 phase
• Fig 2.9
  • End recognition by KU binding
  • Recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)
  • End processing by Artemis (endonuclease)
  • Fill-in synthesis or end bridging by DNA pol μ or λ with Ku/DNA/XRCC4/DNA ligase IV complex
  • Ligation by PNK/XRCC4/DNA ligase IV/XLF complex

Homologous recombination repair (HRR)

1. High fidelity mechanism of repairing DSBs
2. Occurs primarily in the late S/G2 phase
3. Requires physical contact with an undamaged chromatid or chromosome
4. Fig 2.10
   • Damage recognition & DNA resection to form 3’ single strand by MRN (MRE11-Rad50-NBS) complex
   • BRCA1 (phosphorylated by ATM) recruited to the DSBs attract BRCA2
   • Single strand DNA is coated with RPA to form nucleoprotein filament
   • Rad51, Rad52, BRCA1/2 mediates the invasion of the homologous strand of the sister chromatid → Holliday junction
   • Rad54 unwind the double strand
   • Holliday junctions are resolved by noncrossing over or by crossing over
5. Inactivation of HRR results in radiosensitivity and genomic instability
6. Dysregulated HRR lead to cancer by loss of heterozygosity (LOH)

Crosslink repair

• DNA-DNA and DNA-Protein crosslinks by ionizing radiation
• Combination of NER and recombinational repair pathway (Fig 2.11)
• Stalling of the replication fork is the initial signal
• Cells with mutations in NER and HRR pathways are modestly sensitive to crosslinking agent
• Still under investigation

Mismatch repair (MMR)

• Repair base-base and small insertion mismatches during replication & base-base mismatch during HR
• Fig 2.12
  • Recognition of mismatched bases (Msh2-Msh6 or Msh2-Msh3)
  • Recruitment of MMR factors (MLH1-PMS2, MLH1-PMS1, ML1-MLH3) with EXO1
  • Excision of incorrect/altered nucleotides (EXO1)
  • Gap filling by pol δ/ε – RCF – PCNA and ligation
• Mutation in MSH, MLH and PSM families leads to microsatellite instability and cancer.
  E.g. HNPCC

Relationship between DNA Damage and Chromosome Aberrations

• DSBs are the most relevant lesions leading to most biologic insults from radiation including cell killing

Chromosomes and Cell Division

• Interphase: the largest part of the life of any somatic cell, the quantity of DNA doubles
• Mitosis
  • Prophase
    • Thickening of the chromatin and condensation of chromosomes into light coils
    • Centromere by the end of the prophase
    • Nuclear membrane and nucleoli disappear
  • Metaphase
    • Chromosomes move to the center of the cell
    • Spindle forms
  • Anaphase
    • Movement of the chromosomes on the spindle to the poles
  • Telophase
    • Chromosome congregated at the poles of the cell begin to uncoil
    • Nuclear membrane and nucleoli reappear

The Role of Telomeres

• Cap and protect the terminal ends of chromosomes
• Long arrays of TTAGGG repeats → total length 1.5 to 150 kilobases
• Each time a normal cell divides, telomeric DNA is lost from the lagging strand
• After 40-60 divisions, the telomeres in human cells are shortened
  → Vital DNA sequences are lost → No further division, undergoes senescence: “molecular clock”
• Stem cell and cancer cells avoid aging by telomerase
• Telomerase; reverse transcriptase with complementary sequence to TTAGGG repeats and continually rebuilds the chromosome ends
• All human tumor-cell lines and ~90% of human cancer biopsy specimens exhibit telomerase activity

Radiation-Induced Chromosome Aberrations

• X-ray irradiation → DSBs are produced and the broken ends appear to be “sticky”
• Once breaks are produced, different fragments may behave in a variety of ways
  • Breaks rejoin in their original configuration
  • Fail to rejoin and give rise to an aberration
  • Reassort and rejoin to give rise to distorted chromosome
• Aberrations seen at metaphase
  • Chromosome aberrations
    • Early interphase (before duplication)
    • After replication, identical breaks in the corresponding point of a pair of chromatin strands
  • Chromatid aberrations
    • Later interphase (after duplication)
    • Break in a single chromatid arm with the opposite arm of the same chromosome undamaged

Examples of Radiation Induced Aberrations

• Lethal aberrations: gross distortions and visible
  • Dicentric, ring: chromosome aberrations
  • Anaphase bridge: chromatid aberrations
• Non-lethal but carcinogenic aberrations
  • Symmetric translocation
  • Small interstitial deletion
• Dicentric (Fig 2.14A, 2.15B)
  • Interchange between two separate chromosomes
  • Fragment with two centromeres (dicentric) and with no centromere (acentric)
• Ring (Fig 2.14B, 2.15C)
  • Breaks in both arms of the same chromosome
  • Sticky ends rejoin to form a ring and a fragment
• Anaphase bridge (Fig 2.14C, 2.16C)
  • Breaks in both chromatids of the same chromosome
  • Sticky ends rejoin to form a sister union
• Symmetric translocation (Fig 2.17A, 2.18)
  • Breaks in two prereplication (G1) chromosomes à Broken ends exchange
  • Detected with FISH (or chromosome painting)
  • Activation of oncogene, e.g. Burkitt’s lymphoma
• Small interstitial deletion (Fig 2.17B, 2.19)
  • Two breaks in the same arm of the same chromosome
  • Loss of genetic information between the two breaks

Chromosome Aberrations in Human Lymphocytes

• Chromosomal aberrations in peripheral lymphocytes: biomarkers of radiation exposure
• Dose-response curve for aberration in human lymphocytes (Fig 2.20)
  • Fitted by linear-quadratic relationship
    • Linear component – two breaks resulting from single charged particle
    • Quadratic component – two breaks result from different charged particles
• The lowest single dose that can be detected readily is 0.25 Gy
• “Unstable” aberrations
  • Asymmetric aberrations (such as dicentrics) die in attempting subsequent mitosis
  • Their number declines with time after irradiation
• “Stable” aberrations
  • Symmetric nonlethal aberration (such as translocation) survive and pass on the aberration to progeny
  • Persist for many years
• l If many years have elapsed, scoring dicentrics underestimates the dose and only stable aberrations give an accurate picture: FISH