DNA strand break and chromosomal aberration

Girum T.(COR-I)

01-Jun-19 GTZ 1
 GENERAL OVERVIEW OF DNA STRAND BREAKS

– There is strong evidence that DNA is the principal target for the biologic
effects of radiation, including cell killing, carcinogenesis, and mutation.

– A consideration of the biologic effects of radiation, therefore, begins

logically with a description of the breaks in DNA caused by charged-
particle tracks and by the chemical species produced.

– Deoxyribonucleic acid(DNA) is a large molecule with a well-known double

helical structure.

– It consists of two strands held together by hydrogen bonds between the

bases. The “backbone” of each strand consists of alternating sugar and
phosphate groups. The sugar involved is deoxyribose.

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 GENERAL OVERVIEW OF DNA STRAND BREAKS

– Attached to this backbone are four bases, the sequence of which specifies
the genetic code.

– Two of the bases are single-ring groups (pyrimidines); these are thymine
and cytosine.

– Two of the bases are double-ring groups (purines); these are adenine and
guanine.

– The bases on opposite strands must be complementary; adenine pairs

with thymine, and guanine pairs with cytosine.

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 The structure of a single strand of DNA

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– Nucleosides are formed by covalently linking a base to the number
1 carbon of a sugar.

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– Nucleotides are
formed when 1 or
more phosphate
groups is
attached to the
5′carbon of a
nucleoside.

– Nucleoside di- and triphosphates are high-energy compounds

because of the hydrolytic energy associated with the acid
anhydride bonds.

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 – Radiation induces a large number of lesions in DNA, most of which are
repaired successfully by the cell
– A dose of radiation that induces an average of one lethal event per cell
leaves 37% still viable; this is called the D0 dose
– For mammalian cells, the x-ray D0 usually lies between 1 and 2 Gy.

– The number of DNA lesions per cell detected immediately after such a
dose is approximately:
• Base damage, > 1,000
• Single-strand breaks (SSBs), 1,000
• Double-strand breaks (DSBs), 40

– If cells are irradiated with a modest dose of x-rays, many breaks of a single
strand occur. These can be observed and scored as a function of dose if the
DNA is denatured and the supporting structure is stripped away.

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 – In intact DNA, however, SSBs are of little biologic consequence as far as cell
killing is concerned because they are repaired readily using the opposite
strand as a template

– If the repair is incorrect (misrepair), it may result in a mutation.

– If both strands of the DNA are broken and the breaks are well separated ,
repair again occurs readily because the two breaks are handled separately.

– By contrast, if the breaks in the two strands are opposite one another or
separated by only a few base pairs , this may lead to a DSB , resulting in
the cleavage of chromatin into two pieces.

– DSBs are believed to be the most important lesions produced in

chromosomes by radiation; the interaction of two DSBs may result in cell
killing, carcinogenesis, or mutation.

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– Two-dimensional representation of the normal DNA helix. The base pairs
carrying the genetic code are complementary (i.e., adenine pairs with thymine,
guanine pairs with cytosine).

– A break in one strand is of little significance because it is repaired readily using

the opposite strand as a template.

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– Breaks in both strands, if well separated, are repaired as independent breaks

– If breaks occur in both strands and are directly opposite or separated by only a
few base pairs, this may lead to a double-strand break in which the chromatin
snaps into two pieces.

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 – 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,

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 strand break

– The energy from ionizing radiations is not deposited uniformly in the

absorbing medium but is located along the tracks of the charged particles
set in motion—electrons in the case of x- or γ-rays, protons and a-
particles in the case of neutrons.

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 – Radiation chemists speak in terms of “spurs,” “blobs,” and “short tracks.”

– There is, of course, a full spectrum of energy event sizes, and it is quite
arbitrary to divide them into just three categories, but it turns out to be
instructive.

– A spur contains up to 100 eV of energy and involves, on average, three ion

pairs.

– In the case of x- or Y-rays, 95% of the energy deposition events are spurs,
which have a diameter of about 4 nm, which is about twice the diameter
of the DNA double helix

– Blobs are much less frequent for x- or Y-rays; they have a diameter of
about 7 nm and contain on average about 12 ion pairs with an energy
range of 100–500 eV

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 S=small B= Big

• Illustration of a locally multiply damaged site. Energy from x-rays is not absorbed uniformly but tends to be
localized along the tracks of charged particles.
• Radiation chemists speak in terms of spurs and blobs, which contain several ion pairs and have dimensions
comparable to the DNA double helix.
• A double-strand break is likely to be accompanied by extensive base damage. John Ward coined the term
locally multiply damaged site to describe this phenomenon
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 – Because spurs and blobs have dimensions similar to the DNA double helix,
multiple radical attacks occurs if they overlap the DNA helix.

– There is likely to be a wide variety of complex lesions, including base

damage as well as DSBs.

– The term locally multiply damaged site was initially coined by John Ward to
describe this phenomenon, but it has been replaced with the term
clustered lesion. 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.

– In the case of densely ionizing radiations, such as neutrons or a-particles, a

greater proportion of blobs are produced. The damage produced, therefore,
is qualitatively different from that produced by x- or gamma-rays and it is
much more difficult for the cell to repair.

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 Measuring DNA strand breaks

– Over the years, various techniques have been used to measure DNA strand
breaks, including

 sucrose gradient sedimentation

 alkaline and neutral filter elution
 nucleoid sedimentation
 pulsed-field gel electrophoresis (PFGE)
 single-cell gel electrophoresis (also known as the comet assay)

– Of these techniques, PFGE and single-cell gel electrophoresis are still used
to measure DNA strand breaks.

– In addition to these past techniques, radiation

radiation--induced nuclear foci has
become a popular approach to visualize DNA damage through the
recruitment of DNA repair proteins to sites of DNA damage.

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– SSB measurement =alkaline elution
– DSB measurement=neutral comet assay
– Protein detection=western blot
– DNA amplification=PCR
– DNA synthesis= BrdU incorporation assay

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 DNA repair pathways

– Mammalian cells have developed specialized pathways to sense, respond to, and
repair base damage, SSBs, DSBs, sugar damage, and DNA–DNA crosslinks.

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 Base Excision Repair

– Base damage is repaired through the base excision repair (BER) pathway

– Bases on opposite strands of DNA must be complementary; adenine (A)

pairs with thymine (T), and guanine (G) pairs with cytosine (C).

– U therefore represents a putative single-base mutation that is first

removed by a glycosylase/DNA lyase

– Removal of the base is followed by the removal of the sugar residue by an

apurinic endonuclease 1 (APE1), then replacement with the correct
nucleotide by DNA polymerase , and completed by DNA ligase III–XRCC1–
mediated ligation.

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 Base Excision Repair

– If more than one nucleotide is to be replaced (illustrated by the putative

mutation UU, then the complex of replication factor C (RFC)/ proliferating
cell nuclear antigen (PCNA)/DNA polymerase ᵟ/ᵋ performs the repair
synthesis, the overhanging flap structure is removed by the flap
endonuclease 1 (FEN1), and DNA strands are sealed by ligase I

– 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.

– One exception to this is the mutation of the x-ray cross complementing

factor 1 (XRCC1)
XRCC1) gene,
gene which confers about a 1.7-fold increase in radiation
sensitivity. However, the radiation sensitivity of XRCC1-deficient cells may
come from XRCC1’s potential involvement in other repair processes such
as SSBs.

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 Nucleotide Excision Repair

• Nucleotide excision repair (NER) removes bulky adducts in the DNA such as
pyrimidine dimers.

• The process of NER can be subdivided into two pathways:

– Global genome repair (GGR or GG-NER)
– Transcription-coupled repair (TCR or TC-NER)

• The process of GG-NER is genome-wide (i.e., lesions can be removed from DNA
that encodes or does not encode for genes).
• In contrast, TC-NER only removes lesions in the DNA strands of actively transcribed
genes.

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• When a DNA strand that is being actively transcribed becomes damaged, the
RNA polymerase can block access to the site of damage and hence prevents
DNA repair

• TC-NER has evolved to prevent this blockade by RNA polymerase by effectively

removing it from the site of damage to allow the repair proteins access.

• 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

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 Nucleotide excision repair pathways.
• The two sub- pathways of NER, GG-
NER/GGR & TC-NER/TCR , differ at the
initial damage recognition step.

• GGR uses the XPC-XPE protein complexes,

where as in TCR, the NER proteins are
recruited by the stalled RNA polymerase in
cooperation with CSB and CSA.

• Following recognition, the lesion is

demarked by binding of the transcription
factor IIH (TFIIH) complex, XPA and RPA.
• The TFIIH complex helicase function
unwinds the DNA and generates an open
stretch around the lesion, at which point
the XPG and XPF-ERCC1 endonucleases
make incisions at the 3 and 5 ends,
respectively, releasing a 24–32 oligomer.

• The resulting gap is filled by the

polymerases /aided by RFC and PCNA and
the strand is finally ligated.

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• Mutation in NER genes does not lead to ionizing radiation sensitivity.

• However, defective NER increases sensitivity to UV-induced DNA damage and

anticancer agents such as alkylating agents that induce bulky adducts.

• Germline mutations in NER genes lead to human DNA repair deficiency

disorders such as xeroderma pigmentosum in which patients are
hypersensitive to ultraviolet light

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 DNA Double-Strand Break Repair

– In eukaryotic cells, DNA DSBs can be repaired by two basic processes:

• Homologous recombination repair (HRR), which requires an
undamaged DNA strand as a participant in the repair as a template
• Nonhomologous end-joining (NHEJ), which mediates end to-end
joining.

– In lower eukaryotes such as yeast, HRR is the predominant pathway used for
repairing DNA DSBs.

– Homologous recombination is an error-free process because repair is

performed by copying information from the undamaged homologous
chromatid/ chromosome.

– In mammalian cells, the choice of repair is biased by the phase of the cell
cycle and by the abundance of repetitive DNA.
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 – 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, whereas NHEJ occurs in the G1
phase of the cell cycle, when no such template exists

– NHEJ is error prone and probably accounts for many of the premutagenic lesions
induced in the DNA of human cells by ionizing radiation.

– However, it is important to keep in mind that NHEJ and HRR are not mutually
exclusive, and both have been found to be active in the late S/G2phase of the cell
cycle, indicating that other as-yet unidentified factors, in addition to cell cycle
phase, are important in determining what repair program is used.

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 – Illustration showing that nonhomologous recombination occurs in the G1 phase of
the cell cycle, at which stage, there is no sister chromatid to use as a template for
repair.

– In contrast, homologous recombination occurs in the S and G2 phases of the cell

cycle, when there is a sister chromatid to use as a template in repair.
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 Nonhomologous End-Joining
– The immediate response of a cell to a DNA DSB is the activation of a group
of sensors that serve both to promote DNA repair and to prevent the cell
from proceeding in the cell cycle until the break is faithfully repaired.

– These sensors, ATM and Rad3-related (ATR), are protein kinases that
belong to the phosphatidylinositol-3-kinase-related kinase (PIKK) family
and are recruited to the sites of DNA strand breaks induced by ionizing
radiation.

– The competition for repair by HRR versus NHEJ is in part regulated by the
protein 53BP-1.
– Functionally, ATM promotes the processing of broken DNA
ends to generate recombinogenic single-strand DNA by
regulating the activity of the NBS/MRE11/Rad50s(=MRN)
protein complex and this resection activity of ATM is
diminished by 53BP-1
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 – The ligation of DNA DSBs by NHEJ does not require sequence homology.

– However, the damaged ends of DNA DSBs cannot simply be ligated

together; they must first be modified before they can be rejoined by a
ligation reaction.

– 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

– End recognition occurs when the Ku heterodimer, composed of 70-kDa

and 83-kDa subunits, and the DNA-PKcs bind to the ends of the DNA DSB.

– Although the Ku/DNA-PKcs complex is thought to bind ends first, it is still

unknown what holds the two DNA DSB ends together.
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– Although microhomology between one to four nucleotides can aid in end
alignment, there is no absolute requirement for microhomology for NHEJ.

– In fact, Ku does not only recruit DNA-PKcs to the DNA ends, but an additional
protein, Artemis, which possesses endonuclease activity, forms a physical
complex with DNA-PKcs.

– The Ku/DNA-PKcs complex that is bound to the DNA ends can phosphorylate
Artemis and activate its endonuclease activity to deal with 5 and 3 overhangs
as well as hairpins.

– End processing is followed by fill-in synthesis of gaps formed by the Artemis

endonuclease activity.

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– This aspect of NHEJ may not necessarily be essential; for example, in the
ligation of blunt ends or ends with compatible termini.

– At present, it is unclear what the signal is for a fill-in reaction to proceed after
endo nuclease processing.
– However, DNA polymerase or has been found to be associated with the
Ku/DNA/XRCC4/DNA ligase IV complex and serves as the polymerase for the
fill-in reaction.

– In the final step of NHEJ, ligation of nicked DNA ends that have been
processed is mediated by a PNK/XRCC4/DNA ligase IV/XLF complex that is
probably recruited by the Ku heterodimer.

– Polynucleotide kinase (PNK) is a protein that has both 3-DNA phosphatase

and 5-DNA kinase activities and serves to remove end groups that are not
ligatable to allow end joining.

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– NHEJ is error prone and plays an important physiologic role in generating
antibodies through V(D)J rejoining.

– The error-prone nature of NHEJ is essential for generating antibody diversity

and often goes undetected in mammalian cells, as errors in the noncoding
DNA that composes most human genome has little consequence.

– NHEJ is primarily found in the G1 phase of the cell cycle, where there is no
sister chromatid.

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• Nonhomologous endjoining.
• DNA strand breaks are recognized by the ATM and the MRN (MRE11-Rad50-NBS1)
complex, resulting in resection of the DNA ends.
• Homologous recombination is inhibited by the activity of 53BP1.

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• Nonhomologous endjoining. • The initial step of the core NHEJ
pathway starts with the binding
• DNA strand breaks are of the ends at the DSB by the
recognized by the ATM and Ku70/Ku80 heterodimer.
the MRN (MRE11-Rad50- • This complex then recruits and
activates the catalytic subunit of
NBS1) complex, resulting in DNA-PK (DNA-PKcs), whose role
resection of the DNA ends. is the juxtaposition of the two
• Homologous recombination DNA ends.
• The DNA-PK complex then
is inhibited by the activity of recruits the ligase complex
53BP1. (XRCC4/XLF-LIGIV/PNK) that
promotes the final ligation step.

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• The initial step of the
core NHEJ pathway
starts with the binding
of the ends at the DSB
by the Ku70/Ku80
heterodimer.
• This complex then
recruits and activates
the catalytic subunit of
DNA-PK (DNA-PKcs),
whose role is the
juxtaposition of the two
DNA ends.
• The DNA-PK complex
then recruits the ligase
complex (XRCC4/XLF-
LIGIV/PNK) that
promotes the final
ligation step.

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 Homologous Recombination Repair

– HRR provides the mammalian genome a high fidelity mechanism of

repairing DNA DSBs. In particular, the increased activity of this
recombination pathway in late S/G2 suggests that its primary function is
to repair and restore the functionality of replication forks with DNA DSBs.

– Compared to NHEJ, which requires no sequence homology to rejoin

broken ends, HRR requires physical contact with an undamaged chromatid
or chromosome (to serve as a template) for repair to occur.

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 – During recombination, evidence exists that ATM phosphorylates the breast cancer
tumor suppressor protein BRCA1, which is then recruited to the site of the DSB
that has been bound by the NBS/MRE11/Rad 50s(=MRN) protein complex.
– MRE11 and perhaps other yet unidentified endonucleases resect the DNA,
resulting in a 3 single-strand DNA that serves as a binding site for Rad51.

– BRCA2, which is attracted to the DSB by BRCA1, facilitates the loading of Rad51 on
to RPA-coated single-strand overhangs produced by endonuclease resection.

– Rad51 protein is a homologue of the Escherichia coli recombinase RecA and

possesses the ability to form nucleo-filaments and catalyze strand exchange with
the complementary strand in the undamaged chromosome.

– Five additional paralogues of Rad51 also bind to the RPA-coated single-stranded

region and recruit Rad52, which protects against exonucleolytic degradation.

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 – To facilitate repair, Rad54 uses its ATPase activity to unwind the double-stranded
molecule.
– The two invading ends serve as primers for DNA synthesis, and the so-called
Holliday junctions are resolved by MMS4 and MUS81 by noncrossing over, in
which case, the Holliday junctions disengage and DNA strand pairing is followed by
gap filling, or by crossing over of the Holliday junctions, which is followed by gap
filling.

– The identities of the polymerase and ligase involved in these latter steps are
unknown.

– Because inactivation of HRR genes results in radiosensitivity and genomic

instability, these genes provide a critical link between HRR and chromosome
stability.
– Dysregulated homologous recombination can also lead to cancer by loss of
heterozygosity (LOH).

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 The initial step in HR is the recognition of the
lesion and processing of the double-strand
DNA ends into 3DNA single strand tails by the
MRN (Mre11-Rad50-Nbs1) complex, which
are then coated by RPA forming a
nucleoprotein filament.

 Then, specific HR proteins are recruited to the

nucleoprotein filaments, such as RAD51,
RAD52, and BRCA1/2.

 RAD51 is a key protein in homologous

recombination as it mediates the invasion of
the homologous strand of the sister
chromatid, leading to formation of Holliday
junctions.

 The Holliday junctions are finally resolved

into two DNA duplexes.

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 NHEJ Vs HRR

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 Crosslink Repair

– Several DNA–DNA and DNA–protein crosslinks induced by ionizing

radiation have not been extensively studied to arrive at a quantitative
estimate.
– Furthermore, the genes and pathways used for DNA–DNA or DNA–
protein crosslink repair are still under investigation.

– The current thinking is that a combination of NER and recombinational

repair pathways is needed to repair DNA crosslinks

– The predominant signal from a DNA- interstrand crosslink that signals for
repair is stalling of the DNA replication fork.

– The crosslink is removed in a multistep process, first from one strand by a

second round of NER, resulting in a strand break and a DNA adduct.

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 – DNA synthesis can proceed past the lesion, resulting in a point mutation
opposite the lesion. However, the SSB will become a DSB, and seems to
require HRR for restitution. Finally, the adduct that remains is removed by
NER.

– Cells with mutations in NER and HRR pathways are modestly sensitive to
crosslinking agents. In contrast, individuals afflicted with the syndrome
Fanconi anemia are hypersensitive to crosslinking agents.

– Chromatin that contains actively transcribed genes is more susceptible to

DNA–protein crosslinks, and the cross linked proteins are usually nuclear
matrix proteins.

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 DNA–DNA crosslink repair.

 The initial signal for DNA–DNA

crosslinks is stalling of the
replication fork (A).
 The crosslink is removed from
one strand by nucleotide excision
repair (B)
 followed by translesion synthesis,
resulting in a mutation opposite
the adduct (C).
 The resulting DNA double-strand
break is repaired by homologous
recombination (D)
 and the crosslink is removed from
the DNA by another round of
nucleotide excision repair (E–F).

 This schema for crosslink repair is

still a work in progress.

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 Mismatch Repair

• The mismatch repair (MMR) pathway removes base–base and small

insertion mismatches that occur during replication. In addition, the
MMR pathway removes base–base mismatches in homologous
recombination intermediates.

• The process of MMR can be subdivided into four components:

– First, the mismatch must be identified by sensors that transduce

the signal of a mismatched base pair
– Second, MMR factors are recruited
– Third, the newly synthesized strand harboring the mismatch is
identified and the incorrect/altered nucleotides are excised;
– Fourth stage, resynthesis and ligation of the excised DNA tract is
completed.
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– MMR was first characterized in E. coli by the characterization of the
Mutgenes, of which homologues of these gene products have been
identified and extensively characterized in both yeast and humans.

– Mutations in any of the mismatch MSH, MLH, and PSM families of

repair genes leads to microsatellite instability (small base insertions or
deletions) and cancer, especially hereditary nonpolyposis colon cancer
(HNPCC)

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 Mismatch repair.
 The initial step in the mismatch
repair pathway is the recognition of
mismatched bases through either
MSH2-MSH6 OR MSH2-MSH3
complexes.

 These recognition complexes

recruit MLH1-PMS2, MLH1-PMS1,
and MLH1-MLH3, along side the
exonuclease EXO1 that catalyzes
the excision step that follows.

 A gap-filling step by polymerases

δ/ε, RCF, and PCNA is followed by a
final ligation step.

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 Relationship between DNA damage and chromosome aberrations
• Cell killing does not correlate
with SSBs, but relates better
to DSBs.
• Agents (such as hydrogen
peroxide) produce SSBs
efficiently, but very few DSBs,
and also kill very few cells.
• Cells defective in DNA DSB
repair exhibit hypersensitivity
to killing by ionizing radiation
and increased numbers of
chromosome aberrations.
• On the basis of evidence such
as this, it is concluded that
DSBs are the most relevant
lesions leading to most
biologic insults from radiation
including cell killing.
• The reason for this is that
DSBs can lead to chromosomal
aberrations that present
problems at cell division.

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 CHROMOSOMES AND CELL DIVISION

– The backbone of DNA is made of molecules of sugar and phosphates, which

serve as a framework to hold the bases that carry the genetic code.
– Attached to each sugar molecule is a base: thymine, adenine, guanine, or
cytosine.
– This whole configuration is coiled tightly in a double helix.

– The largest part of the life of any somatic cell is spent in interphase, during
which the nucleus, in a stained preparation, appears as a lacework of fine
and lightly stained material in a translucent and colorless material
surrounded by a membrane.

– In the interphase nucleus in most cells, one or more bodies of various sizes
and shapes, called nucleoli,are seen.

– The various events that occur during mitosis are divided into several
phases.
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 Prophase

 The first phase of division is called prophase.

– The beginning of this phase is marked by a thickening of the

chromatin and an increase in its stainability as the chromosomes
condense into light coils.

– By the end of prophase, each chromosome has a lightly staining

constriction known as a centromere; extending from the centromere
are the arms of the chromosome.

– Prophase ends when the chromosomes reach maximal condensation

and the nuclear membrane disappears, as do any nucleoli.

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 Metaphase

– With the disappearance of the nuclear membrane, the nuclear plasm and the
cytoplasm mix.

– Metaphase then follows, in which two events occur simultaneously. The

chromosomes move to the center of the cell (i.e., to the cell’s equator), and the
spindle forms.

– The spindle is composed of fibers that cross the cell, linking its poles. Once the
chromosomes are stabilized at the equator of the cell, their centromeres divide,
and metaphase is complete.

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 Anaphase

– The phase that follows, anaphase, is characterized by a movement of the

chromosomes on the spindle to the poles.

– The chromosomes appear to be pulled toward the poles of the cell by fibers
attached to the centromeres.

– The arms, particularly the long arms, tend to trail behind.

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 Telophase

– Anaphase is followed by the last phase of mitosis, telophase.

– In this phase, the chromosomes, congregated at the poles of the cell, begin
to uncoil.

– The nuclear membrane reappears, as do the nucleoli; and as the phase

progresses, the chromosome coils unwind until the nucleus regains the
appearance characteristic of interphase

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 Meiosis

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 The role of telomeres

– Telomeres cap and protect the terminal ends of chromosomes.

– The name telomere literally means “end part.”

– Mammalian telomeres consist of long arrays of TTAGGG repeats that range

in total length any where from 1.5 to 150 kilobases.

– Each time a normal somatic cell divides, telomeric DNA is lost from the
lagging strand because DNA polymerase cannot synthesize new DNA in
the absence of an RNA primer.

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– Successive divisions lead to progressive shortening, and after 40 to 60
divisions, the telomeres in human cells are shortened dramatically, so that
vital DNA sequences begin to be lost.

– At this point, the cell cannot divide further and undergoes senescence.

– Telomere length has been described as the “molecular clock” or generational

clock because it shortens with age in somatic tissue cells during adult life.

– Stem cells in self- renewing tissues, and cancer cells in particular, avoid this
problem of aging by activating the enzyme telomerase.

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– Telomerase is a reverse transcriptase that includes the complementary
sequence to the TTAGGG repeats and so continually rebuilds the chromosome
ends to offset the degradation that occurs with each division.

– In tissue culture, immortalization of cells—that is, the process whereby cells

pass through a “crisis” and continue to be able to divide beyond the normal
limit—is associated with telomere stabilization and telomerase activity.

– Virtually all human tumor cell lines and approximately 90% of human cancer
biopsy specimens exhibit telomerase activity.

– By contrast, normal human somatic tissues, other than stem cells, do not
possess detectable levels of this enzyme.

– It is an attractive hypothesis that both immortalization and carcinogenesis are

associated with telomerase expression.

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 Radiation-induced chromosome aberrations

– In the traditional study of chromosome aberrations, the effects of

ionizing radiations are described in terms of their appearance when a
preparation is made at the first metaphase after exposure to radiation.

– This is the time when the structure of the chromosomes can be

discerned.

– The study of radiation damage in mammalian cell chromosomes is

hampered by the large number of mammalian chromosomes per cell
and by their small size.

– Most mammalian cells currently available for experimental purposes

have a diploid complement of 40 or more chromosomes.

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– There are exceptions, such as the Chinese hamster, with 22
chromosomes, and various marsupials, such as the rat kangaroo and
woolly opossum, which have chromosome complements of 12 and 14,
respectively.

– Many plant cells, however, contain fewer and generally much larger
chromosomes; consequently, until recently, information on
chromosomal radiation damage accrued principally from studies with
plant cells.

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– If cells are irradiated with x-rays, DSBs are produced in the
chromosomes.

– The broken ends appear to be “sticky” because of unpaired bases and

can rejoin with any other sticky end.

– It would appear, however, that a broken end cannot join with a

normal, unbroken chromosome, although this is controversial.

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– Once breaks are produced, different fragments may behave in various ways:

1. The breaks may restitute, that is, rejoin in their original configuration. In
this case, of course, nothing a miss is visible at the next mitosis.

2. The breaks may fail to rejoin and give rise to an aberration, which is
scored as a deletion at the next mitosis.

3. Broken ends may reassort and rejoin other broken ends to give rise to
chromosomes that appear to be grossly distorted if viewed at the
following mitosis.

– The aberrations seen at metaphase are of two classes:

• Chromosome aberrations
• Chromatid aberrations.

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 Chromosome aberrations

– If a cell is irradiated early in interphase, before the chromosome material has

been duplicated.

– In this case, the radiation-induced break is in a single strand of chromatin;

during the DNA synthetic phase that follows, this strand of chromatin lays
down an identical strand next to itself and replicates the break that has been
produced by the radiation.

– This leads to a chromosome aberration visible at the next mitosis because

there is an identical break in the corresponding points of a pair of chromatin
strands.

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 Chromatid aberrations

– If, on the other hand, the dose of radiation is given later in interphase, after
the DNA material has doubled and the chromosomes consist of two strands
of chromatin, then the aberrations produced are called chromatid
aberrations.

– In regions removed from the centromere, chromatid arms may be fairly well
separated, and it is reasonable to suppose that the radiation might break one
chromatid with out breaking its sister chromatid, or at least not in the same
place.

– A break that occurs in a single chromatid arm after chromosome replication

and leaves the opposite arm of the same chromosome undamaged leads to
chromatid aberrations

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 Examples of radiation-induced aberrations

– Three types of aberrations that are lethal to the cell are described,
followed by two common rearrangements that are consistent with cell
viability but are frequently involved in carcinogenesis.

– The three lethal aberrations are the dicentric; the ring, which are
chromosome aberrations; and the anaphase bridge, which is a
chromatid aberration.

– All three represent gross distortions and are clearly visible.

– Two important types of chromosomal changes that are not lethal to

the cell are symmetric translocations and small deletions.

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 Dicentric
– This aberration involves an interchange between two separate chromosomes.
– If a break is produced in each one early in interphase and the sticky ends are close to one
another, they may rejoin as shown.
– This bizarre interchange is replicated during the DNA synthetic phase, and the result is a
grossly distorted chromosome with two centromeres (hence, dicentric).
– There also are two fragments that have no centromere (acentric fragment), which will
therefore be lost at a subsequent mitosis.

Normal metaphase Dicentric and fragment(arrows).70

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 The steps in the formation of a
dicentric by irradiation of
prereplication (i.e., G1)
chromosomes.
 A break is produced in each of two
separate chromosomes.
 The “sticky” ends may join
incorrectly to form an interchange
between the two chromosomes.
 Replication then occurs in the DNA
synthetic period.
 One chromosome has two
centromeres: a dicentric.
 The acentric fragment will also
replicate and both will be lost at a
subsequent mitosis because,
lacking a centromere, they will not
go to either pole at anaphase

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 Ringis

Ring (arrow)

– A break is induced by radiation in each arm of a single chromatid early in the cell cycle.
– The sticky ends may rejoin to form a ring and a fragment. Later in the cycle, during the
DNA synthetic phase, the chromosome replicates.
– The fragments have no centromere and probably will be lost at mitosis because they will
not be pulled to either pole of the cell.
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 The steps in the formation of a
ring by irradiation of a
prereplication (i.e., G1)
chromosome.

 A break occurs in each arm of

the same chromosome. The
sticky ends rejoin incorrectly to
form a ring and an acentric
fragment.

 Replication then occurs

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 Anaphase bridge

– It results from breaks that occur late in the cell cycle (in G2) after the
chromosomes have replicated. Breaks may occur in both chromatids of
the same chromosome, and the sticky ends may rejoin incorrectly to
form a sister union.

– At anaphase, when the two sets of chromosomes move to opposite

poles, the section of chromatin between the two centromeres is
stretched across the cell between the poles, hindering the separation
into two new progeny cells

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– The two fragments may join but because there is no centromere, the joined
fragments will probably be lost at the first mitosis.

– This type of aberration occurs in human cells and is essentially always lethal.

– It is hard to demonstrate because preparations of human chromosomes usually

are made by accumulating cells at metaphase, and the bridge is only evident
at anaphase.

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 The steps in the formation of an
anaphase bridge by irradiation of a
postreplication (i.e., G2) chromosome.

 Breaks occur in each chromatid of the

same chromosome. Incorrect rejoining
of the sticky ends then occurs in a sister
union.

 At the next anaphase, the acentric

fragment will be lost, one centromere
of the dicentric will go to each pole,
and the chromatid will be stretched
between the poles. Separation of the
progeny cells is not possible; this
aberration is likely to be lethal.

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 Symmetric translocations

– It involves a break in two prereplication (G1) chromosomes, with the broken

ends being exchanged between the two chromosomes
– Translocations are associated with several human malignancies caused by the
activation of an oncogene; Burkitt lymphoma and certain types of leukemia
are examples
 Formation of a symmetric translocation.
– Radiation produces breaks in two
different prereplication chromosomes.
– The broken pieces are exchanged
between the two chromosomes, and the
“sticky” ends rejoin.
– This aberration is not necessarily lethal to
the cell.
– There are examples in which an exchange
aberration of this type leads to the
activation of an oncogene.

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 Small interstitial deletion

– May result from two breaks in the same arm of the same chromosome,
leading to the loss of the genetic information between the two breaks.
– Deletions may be associated with carcinogenesis if the lost genetic material
includes a tumor suppressor gene

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 Formation of a deletion by
ionizing radiation in an
interphase chromosome.
 It is easy to imagine how two
breaks may occur (by a single or
two different charged particles)
in such a way as to isolate a loop
of DNA.
 The “sticky” ends rejoin, and the
deletion is lost at a subsequent
mitosis because it has no
centromere.
 This loss of DNA may include the
loss of a suppressor gene and
lead to a malignant change.

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– The interaction between breaks in different chromosomes is by no
means random(????)

– There is great heterogeneity in the sites at which deletions and

exchanges between different chromosomes occur; for example,
chromosome 8 is particularly sensitive to exchanges.

– Each chromosome is restricted to a domain, and most interactions

occur at the edges of domains, which probably involves the nuclear
matrix.

– Active chromosomes are therefore those with the biggest surface area
to their domains.

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 Chromosome aberrations in human lymphocytes

– Chromosomal aberrations in peripheral lymphocytes have been used widely as

biomarkers of radiation exposure.

– In blood samples obtained for cytogenetic evaluation within a few days to a

few weeks after total body irradiation, the frequency of asymmetric
aberrations (dicentrics and rings) in the lymphocytes reflects the dose
received.

– Lymphocytes in the blood sample are stimulated to divide with a mitogen such
as phytohemagglutinin and are arrested at metaphase, and the incidence of
rings and dicentrics is scored.

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– The dose can be estimated by comparison with in vitro cultures exposed to
known doses.

– A dose response curve for aberrations in human lymphocytes produced by

gamma-rays is fitted by a linear- quadratic relationship, as would be
expected, because (?rings) and dicentrics result from the interaction of two
chromosome breaks

– The linear component is a consequence of the two breaks resulting from a

single charged particle.

– If the two breaks result from different charged particles, the probability of an
interaction is a quadratic function of dose.

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 The frequency of chromosomal
aberrations (dicentrics and rings) is a
linear- quadratic function of dose
because the aberrations are the
consequence of the interaction of
two separate breaks.

 At low doses, both breaks may be

caused by the same electron; the
probability of an exchange
aberration is proportional to dose
(D).

 At higher doses, the two breaks are

more likely to be caused by separate
electrons.

 The probability of an exchange

aberration is proportional to the
square of the dose (D2).
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– If a sufficient number of metaphases are scored, cytogenetic evaluations in
cultured lymphocytes readily can detect a recent total body exposure of as low
as 0.25 Gy in the exposed person.

– Such studies are useful in distinguishing between “real” and “suspected”

exposures, particularly in those instances involving “black film badges” or in
potential accidents in which it is not certain whether individuals who were at
risk for exposure actually received radiation doses.

– Mature T lymphocytes have a finite life span of about 1,500 days and are
eliminated slowly from the peripheral lymphocyte pool. Consequently, the
yield of dicentrics observed in peripheral lymphocytes declines in the months
and years after a radiation exposure.

– During in vivo exposures to ionizing radiation, chromosome aberrations are

induced not only in mature lymphocytes but also in lymphocyte progenitors
in marrow, nodes, or other organs.

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– The stem cells that sustain asymmetric aberrations (such as dicentrics) die
in attempting a subsequent mitosis, but those that sustain a symmetric
nonlethal aberration (such as a translocation) survive and pass on the
aberration to their progeny.

– Consequently, dicentrics are referred to as “unstable” aberrations because

their number declines with time after irradiation.
– Symmetric translocations, by contrast, are referred to as “stable”
aberrations because they persist for many years.

– Either type of aberration can be used to estimate dose soon after

irradiation, but if many years have elapsed, scoring dicentrics
underestimates the dose and only stable aberrations such as
translocations give an accurate picture

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– Until recently, translocations were much more difficult to observe than
dicentrics, but now the technique of FISH makes the scoring of such
symmetric aberrations a relatively simple matter.

– The frequency of translocations assessed in this way correlates with total

body dose in exposed individuals even after more than 50 years, as was
shown in a study of the survivors of the atomic bomb attacks on
Hiroshima and Nagasaki.

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 Summary of pertinent conclusions

– Ionizing radiation induces base damage, SSBs, DSBs, and DNA protein
crosslinks.

– The cell has evolved an intricate series of sensors and pathways to

respond to each type of radiation-induced damage.

– Defective nonhomologous recombination leads to chromosome

aberrations, immune deficiency, and ionizing radiation sensitivity.

– Defective homologous recombination leads to chromatid and

chromosome aberrations, decreased proliferation, and ionizing
radiation sensitivity.

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– There is good reason to believe that DSBs rather than SSBs lead to
important biologic end points including cell death, carcinogenesis, and
mutation.

– Radiation-induced breakage and incorrect rejoining in prereplication

(G1) chromosomes may lead to chromosome aberrations.

– Radiation-induced breakage and incorrect rejoining in post replication

(late S or G2) chromosomes may lead to chromatid aberrations.

– Lethal aberrations include dicentrics, rings, and anaphase bridges.

– Symmetric translocations and small deletions are nonlethal.

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– There is a good correlation between cells killed and cells with
asymmetric exchange aberrations (i.e., dicentrics or rings).

– The incidence of most radiation-induced aberrations is a linear-

quadratic function of dose.

– Scoring aberrations in lymphocytes from peripheral blood may be used

to estimate total body doses in humans accidentally irradiated. The
lowest single dose that can be detected readily is 0.25 Gy.

– Dicentrics are “unstable” aberrations; they are lethal to the cell and
are not passed on to progeny. Consequently, the incidence of
dicentrics declines slowly with time after exposure.

– Translocations are “stable” aberrations; they persist for many years because
they are not lethal to the cell and are passed on to the progeny.
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 References

1. Radiobiology for the radiologist,7th edition

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