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Radiation Targets 1:
DNA, Chromosome and Chromatid
Damage and Repair
Bill McBride
Dept. Radiation Oncology
David Geffen School Medicine
UCLA, Los Angeles, Ca.
[email protected]
WMcB2008
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Objectives:
• Know the limitations of different assays for different
types of DNA strand breaks
• Know the different types of DNA and chromosome
radiation damage
• Understand that multiple DNA repair mechanisms
exist and why
• Be able to discuss repair of base, single strand and
double strand DNA breaks
• Know the molecules involved in homologous
recombination and non-homologous end joining and
how these initiate DNA damage response pathways
• Understand how DNA repair activates the DNA
damage response pathway
• Recognize the role of DNA repair mutations in
carcinogenesis
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• DNA repair enzymes continuously monitor chromosomes to
correct damaged nucleotides
– Endogenous mutagens - ROS from cellular respiration,
hydrolysis, metabolites that act as alkylating agents
– Exogenous mutagens - U.V., cigarette smoke, dietary factors
• Apurinic/Apyrimidinic sites are the most common form of
naturally occurring DNA damage
– 10-20,000 apurinic, 500 apyrimidinic, and 170 8oxyguanines sites produced per day per cell under
physiologic conditions
• The number of DSB/cell/day in vivo are not well known but 510% of dividing mammalian cells in culture have at least 1
chromosome break or chromatid gap
• Each time a cell divides it forms 10DSBs, and 50,000 SS!
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• Failure to repair leads to block in DNA
replication, permanent cell cycle arrest,
senescence, or death
• These are the barriers that prevent
development of genomic instability and
carcinogenesis
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Relevant Properties of DNA
when Measuring Damage
• A very long double-stranded helix with basestacking
• Complementary strands are hybridized to each
other via H-bonding and unwind under alkaline
conditions
• Negatively-charged at physiological pH
• Yield of radiation-induced damage is affected
by macromolecular organization of DNA
From: Watson et al. “Mol. Biol. of the Cell”
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Assays
Popular and classic DNA damage assays
• Neutral and alkaline elution through filters or separation on sucrose gradients - a
classic assay for DSB and SSB+DSB, respectively
• Comet assay - sensitive assay for SSB that can be used for single cells; less
sensitive (10Gy) for DSBs
 H2AX focus formation at DNA DSB - sensitive, currently favored DSB assay
Research DNA damage assays
• DNA unwinding assay - a research assay
• Pulsed field gel electrophoresis - research assay that needs a high radiation dose
• Quantification of damaged bases - a very specific assay, mainly for oxidative
stress
• PCR-based assays - new range of research assays that still require validation
Chromosome/chromatid assays
• Micronucleus formation - classic assay especially in occupational exposure
• Chromosome/ chromatid aberration - classic exposure dosimetric assay
– Conventional staining
– Banding
– FISH
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Neutral and alkaline elution assays
label cells with
tritiated thymidine
2 days
Alkaline/neutral conditions
lyse cells
5%
10%
15%
20%
spin
sucrose gradient
0 Gy
5Gy
10Gy
CPM
Fraction #
ALKALINE CONDITIONS UNWIND DNA AND MEASURES SSB and DSB
NEUTRAL CONDITIONS MEASURES DSB
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% DNA retained
Filter Assay
Neutral elution (pH = 7.4)
Alkaline elution for SSB+DSB (pH = 12.2)
Irradiate cells
Lyse cells on filter
Vacuum elute
Collect eluate and measure DNA concentration
As # of breaks increase, the amount of DNA
eluted through the filter increases
0Gy
5Gy
10Gy
20Gy
Fraction number
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–
Comet Assay
electrophoresis
–
+
DOSE
+
agarose
Lysed cells
• A useful assay because
- It can be automated
- Can be performed on single cells
- Can be performed under neutral or
alkaline conditions to show DSBs and
SSBs, but less sensitive for DSBs
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H2AX Focus Formation
• H2AX is phosphorylated at the site of DNA DSBs
• Antibody to phosphorylated H2AX reveals foci, the number of which
approximates to the number of DSB.
• DNA repair proteins are recruited to the same foci.
• H2AX foci are apparent within a minute; reach a max in 10 min.
Dephosphorylation starts after 30 minutes with a t1/2 of about 2 hr
• The rate of repair and residual damage can be assessed within 24hrs
0Gy
1Gy 2hr
1Gy 24hr
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DNA Unwinding Assay
TIME
This assay is based on the principle of alkaline unwinding of strand
breaks in double-stranded DNA to yield single-stranded DNA with
the number of strand breaks being proportional to the amount of
DNA damage. Breaks are monitored by the fluorescence intensity
of an intercalating dye, such as Hoechst 33258.
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Pulsed-field gel electrophoresis
• Irradiate cells (10 Gy)
and isolate DNA
• Load in gel well
• Run gel with alternating
pulses to force larger
pieces of DNA into the
gel
• Measure amount of
DNA migrating into the
gel by fluorescence or
radiolabel
• As the # of breaks
increase, the amount of
Molecular cut-off @ 10 Mbp
DNA migrating into gel
increases
–/+
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PCR-Based assays
Irradiation
Many qPCR-based assays have been described to
measure DNA breaks and repair. Some introduce
plasmids into cells, others examine in situ genes.
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Micronucleus assay
The micronucleus assay is based on the formation of small membrane bound
DNA fragments i.e. micronuclei. These may originate from acentric fragments
(chromosome fragments lacking a centromere) or whole chromosomes which
are unable to migrate with the rest of the chromosomes during the anaphase
of cell division. Typically, cells are cultured cells with cytochalasin B to induce
metaphase arrest and then stained for DNA.
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Chromosome/Chromatid Aberrations
Cytogenetic damage is normally assessed at first metaphase after
irradiation. The type of cytogenetic damage depends upon where in the
cell cycle the cell is when it is irradiated
Mitosis
Chromosome aberrations
• G1 irradiation
• Both sister chromatids involved
Chromatid aberrations
• S or G2 irradiation
• Usually only 1 chromatid involved
There are 2 basic types of lesion
• Deletion-type
• Exchange-type
G2
G1
S phase
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Deletions
May be stable or unstable
Fragments are lost at mitosis and may form micronuclei
DNA stain
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Exchange-Type Rearrangements
Are of two types:
• Symmetrical (balanced)
gene rearrangements
- Generally stable
- Translocations/Inversions
• Asymmetrical (not balanced)
- Generally lethal
- Dicentrics / Rings
• fail at mitosis
• cell death
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From: Hall
“Radiobiology for the Radiologist”
Chromosomal Aberrations
Intrachromosomal
stable (non-lethal) Pericentric Inversions
non-stable (lethal)
Centric Rings
1
Single break
terminal
deletion
Interchromosomal
Translocations
Dicentrics
2
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1
paracentric
inversion
4
Inter-arm intra-change
pericentric
inversion
2
4
3
Intra-arm intra-change
interstitial
deletion
3
deletion
& ring
Inter-change
translocation
dicentric
& deletion
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Chromatid
Aberrations
3
2
4
1
1
2
Single break
Sister union
terminal
deletion
deletion
& ring
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3
4
Inter-arm intra-change
Inter-change
deletion
& ring
translocation
dicentric
& deletion
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CHROMOSOME ANALYSES
Conventional
Banding
FISH
fluorescence in situ hybridization
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normal
abnormal
Here is an example of a 19 painting probe. The normal 19's are the two right-hand
bright yellow chromosomes. The leftmost bright signal is a portion of chromosome
19 attached to another chromosome. This test was used to confirm the identity of
the extra material as chromosome 19 material.
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Multi-Color FISH in Human Lymphocyte
Chromosomes
Non-irradiated
Irradiated
From: Dr. J.D. Tucker
Multiplex FISH (M-FISH) uses 27 different DNA probes hybridized
simultaneously to human chromosomes. Complex chromosomal
abnormalities can be identified.
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Spectral Karyotyping (SKY) visualizes all 23 pairs of human chromosomes at one time, with
each pair painted in a different fluorescent color. Is used to identify translocations in cancer
cells and genetic abnormalities. SKY involves preparation of a large collection of short
sequences of single-stranded fluorescent DNA probes, each complementary to a unique
region of one chromosome and with a different fluorochrome. The fluorescent probes
essentially paint the set of chromosomes in a rainbow of colors.
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Yield of radiation-induced chromosome
damage
Deletions
Terminal deletion = 1 hit
Chromatid deletion = 1 hit
Interstitial deletion = 2 hits
Yield (Y) ~ linear
Y = k +D
k = background
 = proportionality
DOSE (Gy)
Cornford and Bedford Rad Res 111: 385,1987
Fate:
Deletions lost at mitosis
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Yield of radiation-induced chromosome
damage
Exchange-type “lethal” aberrations
≥ 2 hits required
P (2 hits) = D x D = D2
Y (yield) = k + D2
Y = k + bD2
or 1 hit required
P( 1 hit) = D
Y = D
Y = D + bD2
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A plot of # “lethal” aberrations vs natural log S.F.
showed that an average of 1 lethal lesion
decreased survival by e. In other words,
S.F. = e –(D + bD2)
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DNA Repair
• Classically, there are 2 types
• Sub-Lethal and Potentially Lethal Damage
• These are operationally-defined terms that differ
in the the experimental set up in which they are
demonstrated
– PLDR - single dose
– SLDR - split (fractionated) doses
• The molecular mechanisms may be similar, but
this is not clear
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Potentially Lethal Damage
• Potentially lethal damage is defined as damage that
could cause death, but is modified by post-irradiation
conditions
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Potentially Lethal Damage Repair
IRRADIATE
trypsinize
and plate
at 0 min
trypsinize
and plate
at 15 min
trypsinize
and plate Etc.
at 30 min
S.F.
time (mins)
confluent cells
At about 14 days count colonies
calculate surviving fraction
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Sub-lethal (or accumulated) damage results from accumulation of events
that individually are incapable of killing a cell but that together can be lethal
4 nm
Repairable Sublethal Damage
2 nm
Unrepairable Multiply Damaged Site
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• To account for the time gap between the
production of 2 sublethal lesions (dose rate),
Lea and Catcheside (J Genetics 44:216, 1942)
introduced the factor q
• S.F. = e –(D + qbD2)
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Sublethal Damage Repair
• Assessed by varying the
time between 2 or more
doses of radiation
– Sometimes called
Elkind-type repair
700R
1500R
Repopulation
Redistribution
Repair
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Some Molecular Forms of DNA Repair
•
Base Excision Repair
– Repairs most of the 10-20,000 apurinic and 500 apyrimidinic sites/cell/day that
form spontaneously
– Important for repair of most SSB and base damage after IR.
– Persistence leads to a block in DNA replication, cytotoxic mutations, genetic
instability.
– apurinic/apyrimidinic (AP) endonuclease removes 1-3 nucleotides
– T1/2 <5 mins. Active genes repaired faster than inactive
•
Nucleotide Excision Repair
– Repairs U.V. photodamage, chemical adducts, crosslinks by removing pyrimidine
dimers and other helix distorting lesions. Of minor importance for IR.
– Involved in Global Genome repair and Transcription-Coupled repair
– About 30 nucleotides are excised
•
DNA Mismatch Repair
– Corrects base-base mismatches and small loops
– Important in removing replication errors. Of minor importance for IR.
– Important in connection with hereditary colorectal cancer (hMSH2, hMLH1,
hPMS1, hPMS2) and microsatellite instability
•
Double Strand Break Repair
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•
Enzymes exist that reverse rather than excise DNA
damage exist
–
•
The use of repair molecules and processes depends on
a lot of factors
–
•
eg. MGMT (O6-methylguanine DNA methyltransferase) removes methyl
and other alkyl groups
• “Patients with glioblastoma containing a methylated MGMT
promoter benefited from temozolomide, whereas those who did not
have a methylated MGMT promoter did not have such a benefit.”
Hegi et al NEJM 352:997-1003, 2005
eg. Repair of DNA-DNA cross-links after XRT uses NER
There are about 130 DNA repair genes. Luckily, there
are 3 major molecular processes in common
1. Nucleases remove damaged DNA
2. Polymerases lay down the new structures
3. Ligases restore the phosphodiester backbone
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BER
NER
MMR
DNA N-glycosylases
Recognize and remove damage
AP lyase or endonuclease
Msh2/3 or Msh2/Msh6
Rad14p
Rad1p 5’, Rad2p 3’ incision
Cleave backbone
DNA polymerase Repair patch synthesis
Fills gap
DNA ligase
Ligation
Repair patch synthesis
Ligation
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DSBs
• DSBs can be formed physiologically or pathologically
• Physiological
– During VDJ recombination to form Ab or T cell
receptors
– Class switch breaks to make different Ab isotypes
– Mutations to increase Ab affinity
– During meiosis
• Pathological
– Ionizing radiation
– ROS during cellular respiration
– DNA replication across a nick
– Enzymic action especially at fragile DNA sites
– Topoisomerase failures
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DSB Repair
Recombination Models of DSB Repair #1
• Homologous Recombination
– Uses a sister chromatid (in S and G2) or a second
chromosome (in M) as a template
– Does not occur in G1
– Is relatively error free
– Mutants defective in HR have increased chromosomal
aberrations but can generally repair DSBs (inefficiently)
– The major molecular players are:
• MRN complex, Rad51/Rad52/XRCC2/ Rad54/BRCA1/2
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DSB Repair
• Models of DSB Repair #2
• Non Homologous End Joining uses a non-homologous
template with little or no microhomology
– Imprecise, makes mistakes (an advantage in the immune
system)
– Active at any time in cell cycle
– Efficient at restoring chromosomal integrity
– The major mechanism of DSB repair
– Used physiologically in VDJ rejoining of T cell and Ig receptors
– Mammalian mutants deficient in NHEJ are deficient in DNA
repair and immunity (severe combined immune deficiency scid - in mice and humans)
– The major molecular players are:
• Ku70/Ku80 - Artemis/DNA-PKcs - Cerrunos/XRCC4/ligaseIV
• Microhomology-mediated end joining is an inefficient alternative
that is Ku/ligaseIV independent
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Non Homologous End Joining
•
•
•
•
•
Ku 70/80 (or 86) heterocomplex tethers DNA and recruits DNA-PKcs
that promotes binding of various proteins:
Nucleases that remove damaged DNA
– Artemis/DNA-PKcs bind to form a 5’ to 3’ endonuclease that makes
blunt ends
– DNA-PK is activated on binding DNA
– Autophosphorylation aids binding of other repair proteins
Polymerases that lay down the nucleotide structure
– Pol X family members  and and TdT that have varying degrees of
template dependency.  pol can add nucleotides randomly to
generate microhomology that assists repair
Ligases restore the phosphodiester backbone
– Cernunnos (XLF)/XRCC4/DNA ligase IV complex
– XRCC4/DNA ligase IV are flexible being able to ligate just one
strand or across gaps
Each enzyme has a range of flexibilities, allowing many outcomes
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VDJ rejoining in Ab Formation
Ig L chain
Stem cell
V1
V2
V3
V29
J1
C1
V2
V3 J3
J2
C2
J3
C3
J4
C4
B cell
V1
C3
V29
RAG 1 and RAG 2 endonucleases
make DSB that are re-annealed by
NHEJ to make functional Ig or TCR.
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J4
C4
C2
J1
J2
C1
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N HEJ apparatus
LIGASE IV
XRCC4
Cernunnos
P
P
p
Artemis
PPPPPPPP
KU
70/80
KU
70/80
DNA-PKcs
(catalytic subunit)
PPPPPPPP
DNA-Protein Kinase (DNA-Pk)
phosphorylates
P53, c-jun - apoptosis, etc.
eIF-2 - inhibition of protein synthesis
H2AX - histone phosphorylation
KU 70/80 heterodimer recruits DNA-PKcs, its kinase is activated on binding to DNA
and it autophosphorylates to bind Artemis that processes overhangs to blunt ends.
The Cernunnos/DNA-ligase IV/XRCC4 complex then ligates the DNA.
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DNA-PK
• Only protein known to be activated by binding DSB
• Required for DNA DSB repair and V(D)J rejoining by
NHEJ
• Composed of DNA-PKcs (p450), KU70, Ku80
• A large molecule - 4127 aa, 470kDa, 180 kbp
• Is a ser/thr kinase with homology to PI-3 kinase, but has
no lipid activity.
• Scid mice defective in DNA-PKcs
• Most Scid humans are defective in Artemis, which is
phosphorylated by DNA-PK and binds to DNA DSB to
form an endonuclease
Foci are formed that act as an amplification platform
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Cont
-H2AX foci
function to
stabilize DSB
In DNA-PKcs
cells, they are
more numerous
after irradiation
and persist for
24hrs
XRCC3-ve
DNA-PKcs-ve
0Gy
1Gy
2hr
1Gy
24hr
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DSB Repair
The Mre11/ Rad50/ NBS1 (MRN) Complex is involved as a tether for
DSB for HR
Mre11 has nuclease activity
- NBS = Nijmegen Breakage Syndrome protein (nibrin) binds ATM
- Nijmegen Breakage Syndrome patients are
-
Radiation sensitive
Have microcephaly
Immune deficiencies
Predisposition to lymphoid malignancies
Cells show
- defect in DSB repair
- cell cycle arrest abnormalities
- Including radio-resistant DNA synthesis
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Homologous
Recombination
Involved in stalled replication forks as well as DSB repair
Several complex mechanisms involved
§
M
R
N
Rad 50
ATM -H2AX
MDC1
Rad51
BRCA2
+
+
+
Rad52
DNA polymerases and ligases
dsb
5’ to 3’
DNA polymerase resection
blocked
Strand
invasion
single strand
gap fill
resolution
of Holliday
junction
mis-match repair of
heteroduplex DNA
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• Chromatin structure decondenses at site of
DSB
• Histone acetylation and ubiquitination
involved in DNA repair
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DNA DAMAGE RESPONSE
DNA DSBs
NHEJ
Sensors
Kinases
Ku 70/80
DNA-PK
HR
MRE11, Rad50, Nbs1
ATM
BRCA1
Rad51/52
ATR
Relay
proteins
SIGNAL TRANSDUCTION
Effector
proteins
Cell Cycle Arrest, Apoptosis, DNA repair
DNA DSB repair activates signaling with cellular consequences!
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DNA DAMAGE RESPONSE
UV damage,
Cross-linking agents
DNA DSBs
DNA repair
H2AX
Focus
formation
NHEJ
DNA-Pk
HR
ATM
MRN
ATR
BRCA1/2
Rad51
P*
CHK2
CDC25
p53
mdm2
p53 degradation
p21
CHK1
Bax
Phosphatase
G2 arrest
S phase delay
G1/S arrest apoptosis
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Kinase
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• DNA repair genes are genomic “caretaker” genes
preventing cancer by removing DNA mutations
• Defects in DNA repair genes are very common in
cancers
• Loss of many DNA repair genes is embryonic lethal
or results in genomic instability
• Individuals who are ‘carriers’ of defective DNA
repair genes may be especially sensitive to irradiation
and radiation-induced cancers and may be 5-10% of
the population
– Epidemiological studies have shown that AT
heterozygotes have a predisposition for cancer,
especially for breast cancer in women.
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Autosomal Recessive Disorders with Repair Defects
• Xeroderma pigmentosum (XP) and related Cockayne’s syndrome
–U.V. sensitivity
–At least 7 genes (ERCC 1-6; excision repair cross complementing)
–DNA binding and damage recognition, helicase, endonucleases, transcription
factors, inability to excise dimers
• Fanconi’s anemia
–Mutated in 90% aplastic anemias, commonly in leukemias, 20% solid tumors
–Sensitivity to X-linking agents (e.g. mitomycin C) - genomic instability
–7 genes cloned (A, C, D1, D2, E, F, G); D1 is BRCA2
• Bloom’s and Werner’s syndromes
–Helicases mutated
–Defective recombination and replication
–Cancer predisposition
• Li Fraumeni syndrome
–Rare autosomal dominant
–Breast, soft tissue, bone sarcomas with multiple primaries in childhood
–70% have p53 mutations, others have CHK2 mutations
• Ataxia telangiectasia
• Nijmegen-breakage syndrome
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Ataxia Telengiectasia
•
•
•
•
•
•
•
•
•
•
•
Rare autosomal recessive - 1:20,000-1:1000,000, described in 1920s
70-250 fold excess of leukemia/lymphoma and carcinomas (1960s)
Sensitive to ionizing radiation (1974)
Cerebellar degeneration, progressive ataxia, telangiectasia, immune
deficiency (T and B)
Chromosomal instability, DSB repair defect, initial damage unaltered
Signal transduction defect - low, late p53/ GADD45/ c-jun induction
No G1 arrest, no S phase delay (RDS), G2 arrest altered
AT gene (1995) homology to phosphoinositol-3 kinase superfamily
ATM truncations and missense might give different outcomes
Missense found in 8% of breast cancer patients, 20%CLL. No increase in
truncations.
AT heterozygotes have 1.3-2.9 fold increase in breast cancer risk. No
obvious increase in cytotoxic radiosensitivity.
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Nijmegen Breakage Syndrome
•
•
•
•
•
•
Nibrin (Nbs) gene on chromosome 8q21
Microcephaly, growth and mental retardation
High leukemia risk
Radiation sensitivity
Late, low p53, lack G1/S arrest
Nibrin binds in MRE11, RAD50 (MRN) complex
• AT-Like Disorder (ATLD) is an Mre11 defect
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BRCA1 and BRCA2 Tumor Suppressor Genes
•
•
•
BRCA 1:
– Average 65 % lifetime risk for breast cancer
– 40 percent to 60 percent lifetime risk for second breast cancer (not
reappearance of first tumor)
– Average 39 percent lifetime risk for ovarian cancer
– Increased risk for other cancer types, such as prostate cancer
– BRCA1 cancers tend to be “basal-like”, ER-ve
– Expressed in proliferating cells at G1/S
– Associate with rad51 which is involved in DSB repair in HR
BRCA2 is FANC-D1
– Average 45 % lifetime risk for breast cancer in females, 6% in males
– Average 11 percent lifetime risk for ovarian cancer
– Increased risk for other cancer types, such as pancreatic, prostate,
laryngeal, stomach cancer, and melanoma
Cells with mutated BRCA 1/2 are slightly more sensitive to radiation,
cisplatin and MMC because of their role in homologous recombination
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Human Chromosome Instability and
Radiosensitivity
Syndrome
AT
NBS
ATLD
Li-Fraumeni
Fanconi’s Anemia
Familial Breast Ca
Bloom’s
Werner’s
Lig4
SCID
Gene
ATM
NBS1
MRE11
P53/CHK2
FANA-G
BRCA1/2
BLM helicase
WRN helicase
Ligase4
Artemis
Defect
Sensor
Sensor?
Sensor?
Sensor?
HR
HR
Replication
Replication
NHEJ
NHEJ
XIR
++++
++
++
+/+/+
++
++
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Questions on
Radiation Targets 1:
DNA, Chromosome and Chromatid Damage and Repair
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What is the most common form of DNA
damage existing in cells under normal
conditions?
1. Double strand breaks
2. Apurinic/apyrimidinic sites
3. Interstrand crosslinks
4. Thymidine dimer formation
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What assay would be the most sensitive to measure
radiation-induced DNA double strand breaks?
1. Neutral elution of DNA
2. H2AX focus formation
3. Comet assay under neutral conditions
4. Pulsed field electrophoresis of DNA
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What radiation damage is measured by the alkaline
elution of DNA technique
1. Single strand breaks
2. Base damage
3. Double strand breaks
4. Single and double strand breaks
5. DNA-protein crosslinks
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Which of the following is true for the H2AX focus
formation assay?
1. It measures the ability of radiation to transform
normal cells towards cancer
2. Most foci are seen at 24 hours
3. The foci that form after about 10mins
approximate to the number of radiation-induced
DNA DSBs
4. The foci are dependent of activation of ATM
kinase
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The micronucleus assay
1. Is a measure of DNA damage
2. Measures fragments of nuclei that are lost at
mitosis
3. Is a measure of histone damage
4. Measure micronuclei formed by chromosome
translocations
5. Uses microRNA techniques to measure DNA
damage
WMcB2008
www.radbiol.ucla.edu
Radiation-induced chromosome damage
that is usually lethal is most likely due to
1. Deletions
2. Translocation
3. Exchange-type aberrations
4. Gene loss
5. Dicentics or rings
WMcB2008
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Which of the following is correct about sublethal damage repair. It occurs
1. When cells are held in a non-proliferating
state
2. Between fractions of radiation
3. At the G1/s checkpoint
4. Only at low radiation doses
WMcB2008
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Which of the following repair mechanisms is
most important after X-ray exposure of cells
1. Mismatch repair
2. Nucleotide excision repair
3. Double strand break repair
4. Base excision repair
WMcB2008
www.radbiol.ucla.edu
Double strand breaks are least likely to
contribute to DNA lesions in which of the
following situations
1. Cellular respiration
2. VDJ rejoining to make antibodies
3. Meiosis
4. Antibody class switching
WMcB2008
www.radbiol.ucla.edu
What sets DNA repair of double strand breaks by
homologous recombination apart from non
homologous end joining mechanisms? Its
involvement in
1. G1 cell cycle phase only
2. Cell cycle phases other than G1
3. All cell cycle phases
4. Increasing genomic instability
WMcB2008
www.radbiol.ucla.edu
What sets DNA repair of double strand breaks by
homologous recombination apart from non
homologous end joining mechanisms
1. It does not involve the MRN complex
2. It involves ligases
3. It activates the BRCA tumor suppressor protein
4. It is error-prone
WMcB2008
www.radbiol.ucla.edu
Which of the following is NOT true concerning DNA
protein kinase? It is
1. Critical for DNA DSB repair via the
nonhomologous end joining pathway
2. Formed from Ku proteins and DNA-PK catalytic
subunit
3. Activated on binding DNA DSB
4. Defective in many humans with severe combined
immune deficiency (Scid) disease
5. Defective in scid mice
WMcB2008
www.radbiol.ucla.edu
Which of the following is true about DNA repair
genes
1. They are “gatekeeper” genes that directly
regulate tumor growth by inhibiting growth or by
promoting cell death
2. They are “caretaker” genes that prevent cancercausing mutations
3. There are about 20 in human cells
4. Loss of an individual gene is not commonly a
problem as the system is highly redundant
WMcB2008
www.radbiol.ucla.edu
Which of the following in NOT true about Ataxia
Telengiectasia lymphoblastoid cells
1. They have a D0 of about 50cGy
2. They show a higher than normal level of initial
DNA double strand breaks after exposure to
ionizing irradiation
3. They show no G1 arrest, but normal S phase
arrest after radiation exposure
4. They are slow to elevate p53 following radiation
exposure
WMcB2008
www.radbiol.ucla.edu
Which of the following sets Nijmegan Breakage
Syndrome apart from Ataxia Telengiectasia
1. Cells show less sensitivity to ionizing radiation
2. Patients have no immune deficiency
3. Patients have no neurological problems
4. Cells show normal cell cycle arrest following
irradiation
5. It is part of the MRN complex
WMcB2008
www.radbiol.ucla.edu
Answers
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
NA
2
2
4
3
2
5
2
3
1
2
3
4
3
3
5
WMcB2008
www.radbiol.ucla.edu