DNA damages and repair path of them

1. Molecular Mechanisms of DNA
Damage and Repair
Presented by
F. Ghorbani
4/27/2019
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General Overview of DNA Strand Breaks
OPERATIONAL CLASSIFICATIONS OF RADIATION DAMAGE
DNA Repair Pathways

2. General Overview of DNA Strand Breaks
DNA Structure
Deoxyribonucleic acid (DNA) is a
large molecule with a well-known
double helical structure.
DNA is the principal target for the
biologic effects of radiation,
including cell killing, carcinogenesis,
and mutation.
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3. The “backbone” consists
of alternating sugar and
phosphate groups.
The sugar involved is
deoxyribose. Attached to
this backbone are four
bases, the sequence of
which specifies the
genetic code.
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General Overview of DNA Strand Breaks
DNA Structure

4. Ionizing radiation induces:
1. base damage
2. Single-strand breaks
3. Double-strand breaks
4. DNA protein cross-links
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General Overview of DNA Strand Breaks…

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A: A normal DNA helix.
B: A break in one strand is of little significance.
well separated
Breaks in both strands
directly opposite or separated by only a few base
Diagrams of single- and double-strand DNA breaks caused by radiation

6. D0 dose, How many damage?
A dose of radiation that induces an average of one lethal event
per cell leaves 37% of irradiated cells still viable.
For mammalian cells, the x-ray D0 usually lies between 1-2 Gy.
The number and type of DNA lesions per cell detected
immediately after a dose of 1 Gy of x-rays is approximately:
Double-strand breaks (DSBs)* 40
Single-strand breaks (SSBs)** 1,000
Base damage >2,000
DNA-DNA crosslinks 30
*: The most important lesions produced in chromosomes by radiation
**:If the repair is incorrect (misrepair), it may result in a mutation.
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7. The interaction of two DSBs may result in cell killing,
carcinogenesis, or mutation.
There are many kinds of DSBs, varying in the distance between
the breaks on the two DNA strands and the kinds of end groups
formed.
Their yield in irradiated cells is about 0.04 times that of SSBs, and
they are induced linearly with dose, indicating that they are
formed by single tracks of ionizing radiation.
Both free radicals and direct ionizations may be involved in the
formation of the type of SBs.
“Both free radicals and direct ionizations may be involved
in the formation of the type of strand break illustrated in
Figure 2.2D” Page 58
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8. The interaction of two DSBs
1. The interaction of two DSBs may result in cell killing,
carcinogenesis, or mutation. Page 56
2. To understand why RBE reaches a maximum value in terms of the
production of DSBs because the interaction of two DSBs to form
an exchange-type aberration is the basis of most biologic effects.
Page 207
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9. Spur: for x- or γ-rays: 95% of the energy deposition events are spurs
up to 100 eV of energy
three ion pairs
diameter about 4 nm
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Blobs: less frequent for x or γ-rays
energy range of 100 to 500 eV
12 ion pairs
diameter about 7 nm
What are Spur and Blobs?

10. Spur and Blobs…
Given the size of a spur and the diffusion distance of
hydroxyl free radicals, the clustered lesion could be
spread out up to 20 base pairs.
For neutrons or α-particles, a greater proportion of
blobs are produced.
The damage produced, is different from that produced
by x- or γ-rays.
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11. OPERATIONAL CLASSIFICATIONS OF RADIATION DAMAGE
Radiation damage to mammalian cells can be divided into three
categories:
(1) Lethal damage: irreversible/irreparable and leads irrevocably
to cell death
(2) Potentially lethal damage (PLD): can be modified by post
irradiation environmental conditions
(3) Sublethal damage (SLD): under normal circumstances, can be
repaired in hours unless additional SLD is added, which it can
interact to form lethal damage.
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12. Potentially Lethal Damage Repair(PLD) 4/27/2019
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 The component of radiation damage that is modified by by varying
environmental conditions is known as PLD so cell survival can be
influenced.
 If cells are prevented from dividing for 6 hours or more after irradiation,
PLD repair can occur and survival increases.
Allowing time for the PLD repair
Cell survival is enhanced
 PLD repair is significant for X-rays
but does not occur after neutron
irradiation.

13. It has been suggested that the radioresistance of certain types of
human tumors is linked to their ability to repair PLD.
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Repair of potentially lethal damage in mouse fibrosarcomas
PLD Repair…

14. Sublethal Damage Repair (SLD)
The increasing in cell survival that is observed if a given radiation dose is
split into two fractions separated by a time interval.
Is simply the repair of double-strand breaks.
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Reflects the repair of DNA
breaks before they can
interact to form lethal
chromosomal aberrations.
If a dose is splitted by a time
interval, some double-strand
breaks produced by the first
dose are repaired before the
second dose.

15. SLD Repair…
 Double strand “lethal” damage may be formed by:
(1) single-track (2) multiple-track damage.
The cell killing resulting from single-track damage is the same
whether the dose is a single exposure or fractionated. The same
is not true of multiple-track damage.
SLD repair is significant for x-rays but almost nonexistent
neutrons.
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16. Asynchronous
population
A large dose of
radiation
partly
synchronized
in the S phase
SLD Repair….
Age response function
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Survival of Chinese hamster cells
two fractions of x-rays
incubated at 37° C

17. SLD Repair……
The pattern of repair is a
combination of three processes :
1. The prompt repair of SLD.
2. Reassortment
Progression of cells through
the cell cycle.
3. Repopulation
Increase of surviving fraction
resulting from cell division.
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18. Summary of the repair of SLD 4/27/2019
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19. DNA Repair Pathways
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 DNA in the living cell is subjected to many chemical
alterations.
 The genetic information encoded in the DNA has to
remain uncorrupted.
 Any chemical changes must be corrected.
 A failure to repair DNA produces a mutation.

20. Excision of DNA damage
In these reactions a nucleotide segment containing base
damage, double-helix distortion or mispaired bases is
replaced by the normal nucleotide sequence in a new
DNA polymerase synthesis process.
All of these pathways have been characterized in both
bacterial and eukaryotic organisms.
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i. Base excision repair (BER)
ii. Nucleotide excision repair (NER)
iii. Mismatch repair (MMR)
iv. Strand break repairs.

21. i. Excision of DNA damage
Base Excision Repair (BER)
Bases on opposite strands of DNA must be complementary.
This system recognizes modified bases that are not normally found in DNA.
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BER repairs damage to a single nitrogenous base by deploying enzymes
called glycosylases.
Cytosine to uracil
Adenine to hypoxanthine
Guanine to xanthine
Such bases may be formed by deamination of:

22. BER…
1. When the repair system finds an
unusual base, a glycosylase/DNA
lyase removes it.
2. There are different DNA
glycosylases for different bases.
For example, uracil DNA
glycosylase removes uracil.
3. The nucleotide lacking the base
is removed by an endonuclease;
(AP endonuclease removes the
sugar residue).
4. The gap is filled with the correct
nucleotide by DNA polymerase I.
5. The ends are sealed by DNA
ligase.
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23. i. BER…
U represents a putative single-base mutation that:
1. Removing U by a glycosylase/DNA lyase.
2. Removal of the sugar residue by apurinic
endonuclease 1 (APE1)
3. Replacement with the correct nucleotide by DNA
polymerase β
4. Joined by DNA ligase III–XRCC1–mediated ligation.
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24. ii. BER…
If more than one nucleotide is to be replaced:
1. performing the repair synthesis by the complex of
replication factor C (RFC)/proliferating cell nuclear antigen
(PCNA)/DNA polymerase δ/ε
2. Removing the overhanging flap structure by the flap
endonuclease 1 (FEN1)
3. DNA strands are sealed by ligase.
 Although ionizing radiation–induced base damage is
efficiently repaired, defects in BER may lead to an
increased mutation rate but usually do not result in
cellular radiosensitivity.
 Most common types of DNA damage induced by ionizing
radiation are repaired through base excision repair.
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25. ii. Excision of DNA damage
Nucleotide Excision Repair (NER)
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NER repairs damaged DNA which commonly consists of bulky, helix-
distorting damage, such as pyrimidine dimerization caused by UV light.
Damaged regions are removed in 12–24 nucleotide-long strands in a 3-step
process:
Recognition of damage
Excision of damaged DNA
Resynthesis of removed DNA region.
 This system corrects errors such as:
Formation of thymine dimers
Addition of exogenous chemicals to bases

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Thymine dimers are formed on exposure to UV light.
Two adjacent thymine bases on a strand are bonded together.
Thymine dimer distorts the helix.
The nucleotides in the distorted region are incapable of base pairing.
Thymine dimers are removed by UVr ABC excinuclease
UVr A and UVr B detect the error and unwind the DNA in the region
of the defect.
UVr B nicks the strand a few bases downstream of the defect
UVr C nicks it a few bases upstream
DNA polymerase I adds the correct nucleotides followed by ligation
by DNA ligase
NER…

27. The process of GG-NER is genome-wide (i.e., lesions can be removed
from DNA that encodes or does not encode for genes).
TCNER only removes lesions in the DNA strands of actively transcribed
genes. RNA polymerase can block access to the site of damage and
hence prevents DNA repair. TC-NER remove it from the site of damage to
allow the repair proteins access.
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The process of NER can be subdivided into two pathways:
I. Global Genome Repair (GGR or GG-NER)
II. Transcription-Coupled Repair (TCR or TC-NER).
NER…

28. The mechanism of GG-NER and TC-NER differs only in the detection of
the lesion; the remainder of the pathway used to repair the damage is
the same for both.
The essential steps in this pathway are:
1. Damage recognition
2. DNA incisions that bracket the lesion, usually between 24 and 32
nucleotides in length
3. Removal of the region containing the adducts
4. Repair synthesis to fill in the gap region
5. DNA ligation
Mutation in NER genes, does not lead to ionizing radiation sensitivity but
increases sensitivity to ultraviolet (UV)-induced DNA damage and
anticancer agents such as alkylating agents that induce bulky adducts.
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NER…

29. iii. Excision of DNA damage
Mismatch Repair (MMR)
Mismatch repair corrects errors made when DNA is copied. For
example, a C could be inserted opposite an A, or the polymerase could
slip or stutter and insert two to five extra unpaired bases.
MMR pathway removes base–base and small insertion mismatches
that occur during replication.
MMR system detects a signal of a mismatched base on the new strand,
then corrects the errors that escaped proof-reading.
MMR system has to:
Recognize the newly synthesized strand
Scan the strand rapidly
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30. MMR…
Specific proteins scan the newly synthesized DNA, using adenine
methylation within a GATC sequence as the point of reference.
GATC sequence:
GATC is a palindromic sequence present in DNA.
Adenine is methylated in these sequences.
GATC on the new strand remain unmethylated for some time, this allows
the repair system to recognize the new strand.
GATC sequences act as signposts for mismatch repair system.
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31. MMR…
MutS protein scans the new strand from one GATC to another, Binds to a mispaired
base. MutL proteins binds to MutS.
MutL and MutS activate the GATC endonuclease activity of MutH.
Active MutH nicks the new strand just upstream of the GATC sequence, until the
mispaired nucleotide is removed.
DNA polymerase III adds the correct nucleotides in the gap.
DNA ligase seals the new oligonucleotide with the DNA strand.
Faulty mismatch repair has been linked to HNPCC colon cancer, one of the most
common inherited cancers.
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32. iv. Excision of DNA damage
Repairing Strand Breaks
Ionizing radiation and certain chemicals can produce both single-
strand breaks (SSBs) and double-strand breaks (DSBs) in the DNA
backbone.
A. Single-Strand Breaks (SSBs)
Breaks in a single strand of the DNA molecule are repaired using the
same enzyme systems that are used in Base-Excision Repair (BER).
B. Double-strand breaks (DSBs)
These breaks are extremely deleterious. They disturb replication,
transcription and translation. Double-strand breaks can also result in
chromosomal rearrangements. A number of cancers are caused by
chromosomal rearrangements.
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33. Double-Strand Break Repair
There are two mechanisms to repair a complete break in a DNA
:
Non-Homologous End-Joining (NHEJ): In NHEJ, overhanging
pieces of DNA adjacent to the break are joined.
NHEJ repairs DNA DSBs through the orchestration of end-to-
end joining.
Homologous Recombination Repair (HRR): A sister or
homologous chromosome is used as a template for repair.
HRR is an error-free process, because repair is performed by
copying information from the undamaged homologous
chromatid/chromosome.
The competition for repair by HRR versus NHEJ is in part
regulated by the protein 53BP1.
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In lower eukaryotes such as yeast, HRR is the predominant pathway
used for repairing DNA DSBs.
In mammalian cells, the choice of repair is biased by the phase of the
cell cycle and by the abundance of repetitive DNA.
HRR occurs primarily in the late S/G2 phase of the cell cycle, when an
undamaged sister chromatid is available to act as a template.
NHEJ occurs in the G1 phase of the
cell cycle, when no such template
exists.
NHEJ and HRR are not mutually
exclusive, and both have been
found to be active in the late S/G2
phase of the cell cycle.
NHEJ VS. HRR

35. Non-Homologous End-Joining
1) Promote DNA repair
2) Prevent the cell from proceeding in the cell
cycle until the break is faithfully repaired.
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NHEJ can be divided into five steps:
1. End recognition by Ku binding
2. Recruitment of DNA-dependent protein kinase
catalytic subunit (DNA-PKcs(
3. End processing
4. Fill-in synthesis or end bridging
5. Ligation
The immediate response of a cell to a
DNA DSB is activation of a group of
sensors (ATM and ATR) :

36. Homologous
Recombination Repair
(HRR)
HRR provides the
mammalian genome a high-
fidelity mechanism of
repairing DNA DSBs.
Increased activity of this
recombination pathway in
late S/G2.
HRR requires physical
contact with an undamaged
chromatid or chromosome
for repair .
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37. Crosslink Repair
1. Intra-strand crosslinks:
on one strand of DNA
lead to a block of DNA
polymerase activity and to a
single-strand DNA gap
This gap can be resolved by DNA
polymerase switching to a
template without the intra-strand
crosslink or by translation
synthesis.
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Crosslinks are problematic during
replication and can lead to cell cycle
arrest and even cell death.

38. Crosslink Repair
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2. Inter-strand crosslinks
occur between two strands of DNA
result in a complete block to replication and prevent the
unwinding of duplex DNA, leading to cell death if
unrepaired.
repairing by different protein complexes depending on the
phase of the cell cycle.
 In G1 phase, removing by NER.
Only a small percentage of inter-strand crosslinks are
repaired in G1 phase, and the unrepaired lesions will
present a major problem for the cell in S phase.

39. Thank You
For Your
Attention
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