Measures of Dispersion and Variability: Range, QD, AD and SD
Molecular Mechanisms of DNA and chromosomes damage.pptx
1. MOLECULAR MECHANISMS OF DNA
AND CHROMOSOMES DAMAGE AND
REPAIR
DR. ANIRUDH BHAGAT
DR.PARBHAKAR GUPTA
PG RESIDENTS
DEPT. OF RADIATION
ONCOLOGY
MAMC AND LNH
NEW DELHI 110002
2. OVERVIEW
DNA is the principal target for biologic effects of radiation, incl. cell killing,
carcinogenesis, and mutation.
Breaks in DNA are caused by charged particle tracks and by the chemical species
produced.
Radiation induces large number of DNA lesions.
A dose of radiation that induces an average of one lethal event per cell leaves 37% of
irradiated cells still viable; this is called the D0 dose.
3. TYPES OF DNA DAMAGE
Oxidation, chemotherapy and radiation therapy can all damage DNA. There are many
ways in which this can Occur:
Base Damage: This may cause a point mutation, or it may predispose to
additional DNA damage.
Base Mismatch: A mistake during DNA replication leads to insertion of the wrong
base.
This will cause a point mutation if it is not repaired.
Pyrimidine Dimers: Two adjacent pyrimidine bases are cross-linked by
ultraviolet light. This will cause a point mutation if it is not repaired prior to
replication.
4. Intercalation: This occurs when abnormal chemical groups (such as chemotherapy
drugs) are interposed in the DNA helix. This may prevent gene function and replication.
Crosslinking: This occurs when abnormal chemical bonds are formed within the
DNA molecule, This may prevent gene function and replication, or cause DNA strand
breaks.
Single strand breaks (SSBs): The sugar backbone is broken on one strand but not
the other, This is easily repaired as long as the other strand is still intact
Double strand breaks (DSBs): When the DNA is broken on both strands, it has
"sticky ends" that can react with other DNA strands . This causes chromatid and
chromosome aberrations that may be mutagenic or lethal.
5. IONIZING RADIATION AND DNA DAMAGE
Each Gy of ionizing radiation causes approximately:
• >2000 Base damages
• 1000 SSBs
• 40 DSBs
• 30 DNA-DNA crosslinks
SSBs are of little biologic consequence as far as cell killing is
concerned
The DSB is the primary “mechanism of action” of ionizing radiation. The
number of DSBs correlates with cell killing, the other types of damage do not.
6. Ionizing radiation when travels in a medium it is not deposited uniformly in absorbing
medium, producing spurs, blobs, and short tracks.
7. MECHANISMS OF DOUBLE STRAND BREAKS
Conceptually, there are several ways for lethal damage (DSBs) to occur.
Single hit and Accumulated Intra-track: Damage is done by a single particle.
Accumulated Inter-track: Damage is done by two separate particles, such as two SSBs
combining into a DSB.
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8. ASSAYS FOR DNA DAMAGE
Neutral and alkaline elution
Pulsed field electrophoresis
Comet assay (Single Cell Electrophoresis)
Plasmid based assays
9. CHROMATID AND CHROMOSOME
ABERRATIONS
Aberrations are gross mutations created by double strand breaks.
Chromosome aberrations: An aberration occurs in an unreplicated chromosome.
When the chromosome is replicated, the aberration is identical in both chromatids.
Chromatid aberrations: An aberration occurs in a replicated chromosome, affecting
individual chromatids.
10. STABLE AND UNSTABLE
ABERRATIONS
A stable aberration can persist for years, because it is unlikely to cause cell death.
Stable aberrations do not affect chromosome segregation during mitosis.
Stable aberrations include deletions and symmetrical translocations.
An unstable aberration declines in number over time, because it is highly likely to cause
cell death.
Unstable aberrations prevent chromosomes from properly segregating during mitosis.
Unstable aberrations include dicentrics, rings, and anaphase bridges.
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12. MEASURING DNA DAMAGE
Peripheral Blood Lymphocyte Assay
• Fluorescence in situ hybridization (FISH) can measure unstable and stable aberrations.
Stable aberrations may persist for years.
Total-body radiation doses >0.2 Gy will produce measurable chromosome aberrations
in lymphocytes.
• A linear-quadratic mathematical model can be used to estimate absorbed dose from the
number of aberrations.
Total-body radiation doses >4 Gy cannot be estimated by this technique, because the
lymphocytes undergo rapid apoptosis and disappear.
13. Dose-Response: Linear-Quadratic Curve
Plotting DNA damage versus Absorbed
Dose results in an upward-sloping curve
with a linear-quadratic shape.
Linear damage is directly proportional to
dose, and is measured by the coefficient α.
• This represents single-hit damage and
intra- track accumulated damage, which
are completely independent of fraction size
or dose rate.
Quadratic damage is proportional to dose
squared, and is measured by the
coefficient β.
• This represents inter-track accumulated
damage, which is strongly dependent on
fraction size and dose rate.
This curve is the rationale behind the
linear- quadratic (α/β) model of cell
survival..
DSB production in cells is a linear-quadratic
function of dose. Total DSBs can be expressed
as the sum of “linear” damage (not fraction-size
dependent) and “quadratic” (fraction-size
dependent) damage
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15. DNA REPAIR: Single strand breaks
Base excision repair (BER)
• removes a single damaged base.
• Can only repair very small lesions. Bulky damage must be repaired by other
mechanisms.
Nucleotide excision repair (NER)
• removes the damaged base and several adjacent nucleotides.
• This allows the repair of bulky lesions including UV, alkyl groups, and platinum.
16.
17. DNA REPAIR: Double strand breaks
Homologous recombination repair (HRR)
• HRR is the predominant form of DNA repair during late-S and G2, when sister
chromatids are available.
• A homologous DNA sequence from a sister chromatid is used as a template to restore
the broken DNA sequence.
• This is relatively error-free.
• Can repair DSBs and crosslinks.
• Molecules involved in this pathway include rad52, the rad51 complex,
BLM/WRN/RECQ4, and BRCA1/BRCA2.
• BRCA1/2 mutations are responsible for hereditary breast and ovarian cancer syndrome.
18. Non-Homologous End Joining (NHEJ)
• NHEJ is the predominant form of DNA repair during G0/G1, as no sister chromatids exist.
Can also occur in S andG2 phases, although HRR is preferred.
• Error-prone, may lead to mutation or cell death.
• Two broken ends are joined together in a process that may delete some of the DNA near
the junction.
• Molecules involved in this pathway include Ku70/80, Artemis, DNA-PK, XRCC 4, and
Ligase 4.
• DSBs are the main mechanism of radiation-induced cell killing, so any defect in DSB
repair can increase radiation sensitivity.
20. MISMATCH REPAIR
• This procedure removes base-base and small insertion
mismatches that occur during replication
• This constitutes four steps
– Mismatch identification by sensors
– MMR factors are recruited
– Incorrect/altered nucleotide are excised
– Resynthesis and ligation of the excised DNA tract
21. CROSSLINK REPAIR
• Radiation damage produces DNA-DNA and DNA-protein
crosslinks
• Repair mechanism of this crosslinks still under investigation
• Probably combination of HRR and NER are needed for
repair
• Individuals with Fanconi’s anemia are hypersensitive to
croslinking
22. DNA REPAIR MECHANISM DEFECTS
NER disorders
• Xeroderma pigmentosum (XP- gene family)
Photosensitive and very high skin cancer risk.
UV hyper-sensitive, not radiosensitive.
• Cockayne Syndrome (CSA/CSB)
Photosensitive but no cancer risk.
UV hyper-sensitive, not radiosensitive.
• Trichothiodystrophy
23. MMR disorders
• Lynch Syndrome (MLH/MSH gene family).
Extremely high risk of colorectal cancer.
Not radiosensitive, but may be hyper-sensitive to chemotherapy.
HRR disorders
• Hereditary Breast and Ovarian Cancer Syndrome (BRCA1/BRCA2)
Despite the DNA repair defect, BRCA1/2 patients are not extremely radiosensitive
NHEJ
• Ataxia telengectesia
• Imuune deficiency
24. Chromosomal aberrations in peripheral lymphocytes have been used as
biomarkers of radiation exposure.
Frequency of asymmetric aberrations (dicentrics and rings) in the
lymphocytes reflects the dose received.
Dose can be estimated by comparison with in vitro cultures exposed to known
doses.
Lethal aberrations are referred to as “unstable” aberrations as their number
declines with time.
Symmetric translocations are referred to as “stable” aberrations because they
persist for many years.
