objectives: know the limitations of different assays for different types of dna strand breaks

www.rwww.radbioladbiol.ucla.edu.ucla.edu

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Radiation Targets 1: Radiation Targets 1: DNA, Chromosome and Chromatid DNA, Chromosome and Chromatid

Damage and RepairDamage and Repair

Bill McBrideBill McBrideDept. Radiation OncologyDept. Radiation Oncology

David Geffen School MedicineDavid Geffen School MedicineUCLA, Los Angeles, Ca.UCLA, Los Angeles, Ca.

[email protected]@mednet.ucla.edu


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Objectives:Objectives:• Know the limitations of different assays for different types of Know the limitations of different assays for different types of

DNA strand breaksDNA strand breaks• Know the different types of DNA and chromosome radiation Know the different types of DNA and chromosome radiation

damagedamage• Understand that multiple DNA repair mechanisms exist and Understand that multiple DNA repair mechanisms exist and

whywhy• Be able to discuss repair of base, single strand and double Be able to discuss repair of base, single strand and double

strand DNA breaksstrand DNA breaks• Know the molecules involved in Know the molecules involved in homologous recombinationhomologous recombination

and and non-homologous end joiningnon-homologous end joining and how these initiate DNA and how these initiate DNA damage response pathwaysdamage response pathways

• Understand how DNA repair activates the DNA damage Understand how DNA repair activates the DNA damage response pathwayresponse pathway

• Recognize the role of DNA repair mutations in carcinogenesis Recognize the role of DNA repair mutations in carcinogenesis


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• DNA repair enzymes continuously monitor chromosomes to DNA repair enzymes continuously monitor chromosomes to correct damaged nucleotidescorrect damaged nucleotides– Endogenous mutagens - ROS from cellular respiration, Endogenous mutagens - ROS from cellular respiration,

hydrolysis, metabolites that act as alkylating agentshydrolysis, metabolites that act as alkylating agents– Exogenous mutagens - U.V., cigarette smoke, dietary factorsExogenous mutagens - U.V., cigarette smoke, dietary factors

• Apurinic/Apyrimidinic sites are the most common form of Apurinic/Apyrimidinic sites are the most common form of naturally occurring DNA damagenaturally occurring DNA damage– 10-20,000 apurinic, 500 apyrimidinic, and 170 8-10-20,000 apurinic, 500 apyrimidinic, and 170 8-

oxyguanines sites produced per day per cell under oxyguanines sites produced per day per cell under physiologic conditionsphysiologic conditions

• The number of DSB/cell/day in vivo are not well known but 5-The number of DSB/cell/day in vivo are not well known but 5-10% of dividing mammalian cells in culture have at least 1 10% of dividing mammalian cells in culture have at least 1 chromosome break or chromatid gapchromosome break or chromatid gap

• Each time a cell divides it forms 10DSBs, and 50,000 SS!Each time a cell divides it forms 10DSBs, and 50,000 SS!


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• Failure to repair leads to block in DNA Failure to repair leads to block in DNA replication, permanent cell cycle arrest, replication, permanent cell cycle arrest, senescence, or deathsenescence, or death

• These are the barriers that prevent These are the barriers that prevent development of genomic instability and development of genomic instability and carcinogenesis carcinogenesis


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• A very long double-stranded helix with base-stacking

• 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

Relevant Properties of DNARelevant Properties of DNAwhen Measuring Damage when Measuring Damage

From: Watson et al. “Mol. Biol. of the Cell”


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Popular and classic DNA damage assaysPopular and classic DNA damage assays• Neutral and alkaline elution through filters or separation on sucrose gradients - Neutral and alkaline elution through filters or separation on sucrose gradients - a a

classic assay for DSB and SSB+DSB, respectivelyclassic assay for DSB and SSB+DSB, respectively • Comet assay - Comet assay - sensitive assay for SSB that can be used for single cells; less sensitive assay for SSB that can be used for single cells; less

sensitive (10Gy) for DSBssensitive (10Gy) for DSBs H2AX focus formation at DNA DSB - H2AX focus formation at DNA DSB - sensitive, currently favored DSB assaysensitive, currently favored DSB assay

Research DNA damage assaysResearch DNA damage assays• DNA unwinding assay - DNA unwinding assay - a research assaya research assay• Pulsed field gel electrophoresis - Pulsed field gel electrophoresis - research assay that needs a high radiation doseresearch assay that needs a high radiation dose• Quantification of damaged bases - Quantification of damaged bases - a very specific assay, mainly for oxidative stress a very specific assay, mainly for oxidative stress • PCR-based assays - PCR-based assays - new range of research assays that still require validationnew range of research assays that still require validation

Chromosome/chromatid assaysChromosome/chromatid assays• Micronucleus formation - Micronucleus formation - classic assay especially in occupational exposureclassic assay especially in occupational exposure • Chromosome/ chromatid aberration - Chromosome/ chromatid aberration - classic exposure dosimetric assayclassic exposure dosimetric assay

– Conventional stainingConventional staining– BandingBanding– FISHFISH

AssaysAssays


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sucrose gradient

Neutral and alkaline elution assaysNeutral and alkaline elution assays

label cells withtritiated thymidine

2 days lyse cells

5%10%15%20%

spin

ALKALINE CONDITIONS UNWIND DNA AND MEASURES SSB and DSBNEUTRAL CONDITIONS MEASURES DSB

CPM

0 Gy 5Gy 10Gy

Fraction #

Alkaline/neutral conditionsAlkaline/neutral conditions


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Fraction numberFraction number

% D

NA

re

tain

ed

% D

NA

re

tain

ed 0Gy0Gy

5Gy5Gy

10Gy10Gy

20Gy20Gy

Neutral elution (pH = 7.4) Neutral elution (pH = 7.4) Alkaline elution for SSB+DSB (pH = 12.2)Alkaline elution for SSB+DSB (pH = 12.2)Irradiate cellsIrradiate cellsLyse cells on filterLyse cells on filterVacuum eluteVacuum eluteCollect eluate and measure DNA concentrationCollect eluate and measure DNA concentration

As # of breaks increase, the amount of DNA As # of breaks increase, the amount of DNA eluted through the filter increaseseluted through the filter increases

Filter AssayFilter Assay


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CometComet Assay Assay

DOSEelectrophoresiselectrophoresis

agaroseagarose

– – ++

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 is phosphorylated at the site of DNA DSBsH2AX is phosphorylated at the site of DNA DSBs

• Antibody to phosphorylated Antibody to phosphorylated H2AX reveals foci, the number of which H2AX reveals foci, the number of which approximates to the number of DSB.approximates to the number of DSB.

• DNA repair proteins are recruited to the same foci.DNA repair proteins are recruited to the same foci.

• H2AX foci are apparent within a minute; reach a max in 10 min. H2AX foci are apparent within a minute; reach a max in 10 min. Dephosphorylation starts after 30 minutes with a tDephosphorylation starts after 30 minutes with a t1/21/2 of about 2 hr of about 2 hr

• The rate of repair and residual damage can be assessed within 24hrsThe rate of repair and residual damage can be assessed within 24hrs

H2AX Focus FormationH2AX Focus Formation

0Gy0Gy 1Gy 2hr1Gy 2hr 1Gy 24hr1Gy 24hr


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DNA Unwinding AssayDNA Unwinding Assay

TIMETIME

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 electrophoresisPulsed-field gel electrophoresis

– / +

Molecular cut-off @ 10 Mbp

•Irradiate cells (10 Gy) Irradiate cells (10 Gy) and isolate DNAand isolate DNA•Load in gel wellLoad in gel well•Run gel with Run gel with

alternating pulses to alternating pulses to force larger pieces of force larger pieces of DNA into the gelDNA into the gel•Measure amount of Measure amount of

DNA migrating into the DNA migrating into the gel by fluorescence or gel by fluorescence or radiolabelradiolabel•As the # of breaks As the # of breaks

increase, the amount of increase, the amount of DNA migrating into gel DNA migrating into gel increasesincreases


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PCR-Based assaysPCR-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 assayMicronucleus 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 Typically, cells are cultured cells with cytochalasin B to induce metaphase arrest and then stained for DNA.metaphase arrest and then stained for DNA.


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Cytogenetic damage is normally assessed at first metaphase after Cytogenetic damage is normally assessed at first metaphase after irradiation. The type of cytogenetic damage depends upon where in the irradiation. The type of cytogenetic damage depends upon where in the cell cycle the cell is when it is irradiatedcell cycle the cell is when it is irradiated

Chromosome aberrationsChromosome aberrations• GG11 irradiation irradiation• Both sister chromatids involvedBoth sister chromatids involved

Chromatid aberrationsChromatid aberrations• S or GS or G22 irradiation irradiation• Usually only 1 chromatid involved Usually only 1 chromatid involved

Chromosome/Chromatid Chromosome/Chromatid AberrationsAberrations

There are 2 basic types of lesionThere are 2 basic types of lesion • Deletion-typeDeletion-type• Exchange-typeExchange-type

Mitosis

G1

G2

S phase


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DeletionsDeletions

DNA stain

May be stable or unstableMay be stable or unstableFragments are lost at mitosis and may form micronucleiFragments are lost at mitosis and may form micronuclei


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Exchange-Type RearrangementsExchange-Type Rearrangements

From: Hall From: Hall ““Radiobiology for the RadiologistRadiobiology for the Radiologist””

Are of two types:Are of two types:•Symmetrical (balanced)Symmetrical (balanced)gene rearrangementsgene rearrangements-Generally stableGenerally stable-Translocations/InversionsTranslocations/Inversions

•Asymmetrical (not balanced)Asymmetrical (not balanced)-Generally lethalGenerally lethal-Dicentrics / RingsDicentrics / Rings•fail at mitosisfail at mitosis•cell deathcell death


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Single break Intra-arm intra-change Inter-arm intra-change Inter-changeSingle break Intra-arm intra-change Inter-arm intra-change Inter-change

1 2 3 41 2 3 4

terminal interstitial paracentric pericentric deletion translocation dicentricterminal interstitial paracentric pericentric deletion translocation dicentricdeletion deletion inversion inversion & ring & deletiondeletion deletion inversion inversion & ring & deletion

Chromosomal AberrationsChromosomal AberrationsIntrachromosomalIntrachromosomal InterchromosomalInterchromosomal

stable (non-lethal)stable (non-lethal) Pericentric Inversions Translocations Pericentric Inversions Translocationsnon-stable (lethal)non-stable (lethal) Centric Rings Centric Rings DicentricsDicentrics 11 22

33

44


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Single break Sister union Inter-arm intra-change Inter-changeSingle break Sister union Inter-arm intra-change Inter-change

1 2 3 41 2 3 4

terminal deletion deletion translocation dicentricterminal deletion deletion translocation dicentricdeletion & ring & ring & deletion deletion & ring & ring & deletion

ChromatidChromatidAberrationsAberrations

11

22 3344


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Conventional

Banding

FISH fluorescence in situ hybridization fluorescence in situ hybridization

CHROMOSOME ANALYSESCHROMOSOME ANALYSES


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Here is an example of a 19 painting probe. The normal 19's are the two right-hand 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 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 19 attached to another chromosome. This test was used to confirm the identity of the extra material as chromosome 19 material.the extra material as chromosome 19 material.

normal

abnormal


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Multi-Color FISH in Human Lymphocyte Multi-Color FISH in Human Lymphocyte ChromosomesChromosomes

Non-irradiatedNon-irradiated IrradiatedIrradiated

From: Dr. J.D. Tucker

Multiplex FISH (M-FISH) uses 27 different DNA probes hybridized Multiplex FISH (M-FISH) uses 27 different DNA probes hybridized simultaneously to human chromosomes. Complex chromosomal simultaneously to human chromosomes. Complex chromosomal abnormalities can be identified. abnormalities can be identified.


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Spectral Karyotyping (SKY) visualizes all 23 pairs of human chromosomes at one time, with 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 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 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 sequences of single-stranded fluorescent DNA probes, each complementary to a unique region of one chromosome and with a different fluorochrome. The fluorescent probes region of one chromosome and with a different fluorochrome. The fluorescent probes essentially paint the set of chromosomes in a rainbow of colors.essentially paint the set of chromosomes in a rainbow of colors.


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Yield of radiation-induced Yield of radiation-induced chromosome damagechromosome damage

DOSE (Gy)DOSE (Gy)

DeletionsDeletionsTerminal deletion = 1 hitTerminal deletion = 1 hitChromatid deletion = 1 hitChromatid deletion = 1 hitInterstitial deletion = 2 hitsInterstitial deletion = 2 hits

Yield (Y) ~ linearYield (Y) ~ linear

Y = k +Y = k +DD

k = backgroundk = background = proportionality= proportionality

Fate:Fate:Deletions lost at mitosisDeletions lost at mitosis

Cornford and Bedford Rad Res 111: 385,1987


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Yield of radiation-induced Yield of radiation-induced chromosome damagechromosome damage

Exchange-type “lethal” aberrationsExchange-type “lethal” aberrations≥ ≥ 2 hits required2 hits required or 1 hit requiredor 1 hit required

P (2 hits) = D x D = DP (2 hits) = D x D = D22

Y (yield) = k + DY (yield) = k + D22

Y = k + Y = k + DD22

P( 1 hit) = DP( 1 hit) = DY = Y = DD

Y = Y = D + D + DD22


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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 S.F. = e –(–(D + D + D2)D2)


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DNA RepairDNA Repair

• Classically, there are 2 typesClassically, there are 2 types• Sub-Lethal and Potentially Lethal Damage Sub-Lethal and Potentially Lethal Damage • These are These are operationally-definedoperationally-defined terms that differ terms that differ

in the the experimental set up in which they are in the the experimental set up in which they are demonstrateddemonstrated– PLDR - single dosePLDR - single dose– SLDR - split (fractionated) dosesSLDR - split (fractionated) doses

• The molecular mechanisms may be similar, but The molecular mechanisms may be similar, but this is not clear this is not clear


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Potentially Lethal DamagePotentially Lethal Damage

• Potentially lethal damage is defined as damage that Potentially lethal damage is defined as damage that could cause death, but is modified by could cause death, but is modified by post-irradiation post-irradiation conditionsconditions


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IRRADIATEIRRADIATE

At about 14 days count coloniesAt about 14 days count coloniescalculate surviving fractioncalculate surviving fraction

confluent cellsconfluent cells

trypsinizetrypsinizeand plateand plate at 0 minat 0 min



Etc.Etc.

time (mins)time (mins)

S.F.S.F.

Potentially Lethal Damage RepairPotentially Lethal Damage Repair


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Repairable Sublethal DamageRepairable Sublethal Damage

Sub-lethal (or accumulated) damageSub-lethal (or accumulated) damage results from accumulation of events results from accumulation of events that individually are incapable of killing a cell but that together can be lethalthat individually are incapable of killing a cell but that together can be lethal

4 nm4 nm

2 nm2 nm

Unrepairable Multiply Damaged SiteUnrepairable Multiply Damaged Site


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• To account for the time gap between the To account for the time gap between the production of 2 sublethal lesions (dose rate), production of 2 sublethal lesions (dose rate), Lea and Catcheside (J Genetics 44:216, 1942) Lea and Catcheside (J Genetics 44:216, 1942) introduced the factor qintroduced the factor q

• S.F. = e S.F. = e –(–(D + qD + qD2)D2)


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RedistributionRedistribution

RepairRepair

RepopulationRepopulation

700R 1500R

Sublethal Damage RepairSublethal Damage Repair

• Assessed by varying the Assessed by varying the time between 2 or more time between 2 or more doses of radiationdoses of radiation– Sometimes called Sometimes called

Elkind-type repairElkind-type repair


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Some Molecular Forms of DNA RepairSome Molecular Forms of DNA Repair• Base Excision RepairBase Excision Repair

– Repairs most of the 10-20,000 apurinic and 500 apyrimidinic sites/cell/day that form Repairs most of the 10-20,000 apurinic and 500 apyrimidinic sites/cell/day that form spontaneously spontaneously

– Important for repair of most SSB and base damage after IR.Important for repair of most SSB and base damage after IR.– Persistence leads to a block in DNA replication, cytotoxic mutations, genetic instability.Persistence leads to a block in DNA replication, cytotoxic mutations, genetic instability.– apurinic/apyrimidinic (AP) endonucleaseapurinic/apyrimidinic (AP) endonuclease removes 1-3 nucleotides removes 1-3 nucleotides– T1/2 <5 mins. Active genes repaired faster than inactiveT1/2 <5 mins. Active genes repaired faster than inactive

• Nucleotide Excision RepairNucleotide Excision Repair – Repairs U.V. photodamage, chemical adducts, crosslinks by removing pyrimidine Repairs U.V. photodamage, chemical adducts, crosslinks by removing pyrimidine

dimers and other helix distorting lesions. Of minor importance for IR.dimers and other helix distorting lesions. Of minor importance for IR.– Involved in Global Genome repair and Transcription-Coupled repairInvolved in Global Genome repair and Transcription-Coupled repair– About 30 nucleotides are excisedAbout 30 nucleotides are excised

• DNA Mismatch RepairDNA Mismatch Repair – Corrects base-base mismatches and small loopsCorrects base-base mismatches and small loops– Important in removing replication errors. Of minor importance for IR.Important in removing replication errors. Of minor importance for IR.– Important in connection with hereditary colorectal cancer (hMSH2, hMLH1, hPMS1, Important in connection with hereditary colorectal cancer (hMSH2, hMLH1, hPMS1,

hPMS2) and microsatellite instabilityhPMS2) and microsatellite instability• Double Strand Break RepairDouble Strand Break Repair


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• Enzymes exist that reverse rather than excise DNA Enzymes exist that reverse rather than excise DNA damage existdamage exist– eg. MGMT (Oeg. MGMT (O66-methylguanine DNA-methylguanine DNA methyltransferase) removes methyl methyltransferase) removes methyl

and other alkyl groupsand other alkyl groups• ““Patients with glioblastoma containing a methylatedPatients with glioblastoma containing a methylated MGMTMGMT

promoter benefited from temozolomide, whereas those whopromoter benefited from temozolomide, whereas those who did not did not have a methylated have a methylated MGMTMGMT promoter did not have such a promoter did not have such a benefit.benefit.””

Hegi et al NEJM Hegi et al NEJM 352:997-1003, 2005352:997-1003, 2005

• The use of repair molecules and processes depends on The use of repair molecules and processes depends on a lot of factorsa lot of factors– eg. Repair of DNA-DNA cross-links after XRT uses NEReg. Repair of DNA-DNA cross-links after XRT uses NER

• There are about 130 DNA repair genes. Luckily, there There are about 130 DNA repair genes. Luckily, there are 3 major molecular processes in commonare 3 major molecular processes in common– Nucleases remove damaged DNANucleases remove damaged DNA– Polymerases lay down the new structuresPolymerases lay down the new structures– Ligases restore the phosphodiester backboneLigases restore the phosphodiester backbone


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BERBER NER NER MMR MMR

LigationLigation

Repair patch synthesisRepair patch synthesis

LigationLigation

DNA N-glycosylasesDNA N-glycosylasesRecognize and remove damageRecognize and remove damage

AP lyase or endonucleaseAP lyase or endonuclease

Cleave backboneCleave backbone

DNA polymeraseDNA polymeraseFills gapFills gap

Repair patch synthesisRepair patch synthesis

DNA ligaseDNA ligase

Rad14p

Rad1p 5’, Rad2p 3’ incision

Msh2/3 or Msh2/Msh6


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DSBsDSBs• DSBs can be formed physiologically or pathologicallyDSBs can be formed physiologically or pathologically• Physiological Physiological

– During VDJ recombination to form Ab or T cell During VDJ recombination to form Ab or T cell receptorsreceptors

– Class switch breaks to make different Ab isotypesClass switch breaks to make different Ab isotypes– Mutations to increase Ab affinityMutations to increase Ab affinity– During meiosisDuring meiosis

• PathologicalPathological– Ionizing radiationIonizing radiation– ROS during cellular respirationROS during cellular respiration– DNA replication across a nickDNA replication across a nick– Enzymic action especially at fragile DNA sitesEnzymic action especially at fragile DNA sites– Topoisomerase failuresTopoisomerase failures


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DSB RepairDSB Repair

Recombination Models of DSB Repair #1Recombination Models of DSB Repair #1• Homologous RecombinationHomologous Recombination

– Uses a sister chromatid (in S and G2) or a second Uses a sister chromatid (in S and G2) or a second chromosome (in M) as a templatechromosome (in M) as a template

– Does not occur in G1Does not occur in G1– Is relatively error freeIs relatively error free

– Mutants defective in HR have increased chromosomal Mutants defective in HR have increased chromosomal aberrations but can generally repair DSBs (inefficiently)aberrations but can generally repair DSBs (inefficiently)

– The major molecular players are:The major molecular players are:• MRN complex, Rad51/Rad52/XRCC2/ Rad54/BRCA1/2MRN complex, Rad51/Rad52/XRCC2/ Rad54/BRCA1/2


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• Models of DSB Repair #2Models of DSB Repair #2

• Non Homologous End Joining uses a non-homologous Non Homologous End Joining uses a non-homologous template with little or no microhomology template with little or no microhomology – Imprecise, makes mistakes (an advantage in the immune Imprecise, makes mistakes (an advantage in the immune

system)system)– Active at any time in cell cycleActive at any time in cell cycle– Efficient at restoring chromosomal integrityEfficient at restoring chromosomal integrity– The major mechanism of DSB repairThe major mechanism of DSB repair– Used physiologically in VDJ rejoining of T cell and Ig receptorsUsed physiologically in VDJ rejoining of T cell and Ig receptors– Mammalian mutants deficient in NHEJ are deficient in DNA Mammalian mutants deficient in NHEJ are deficient in DNA

repair and immunity (severe combined immune deficiency - repair and immunity (severe combined immune deficiency - scid - in mice and humans)scid - in mice and humans)

– The major molecular players are:The major molecular players are:• Ku70/Ku80 - Artemis/DNA-PKcs - Cerrunos/XRCC4/ligaseIVKu70/Ku80 - Artemis/DNA-PKcs - Cerrunos/XRCC4/ligaseIV• Microhomology-mediated end joining is an inefficient alternative Microhomology-mediated end joining is an inefficient alternative

that is Ku/ligaseIV independentthat is Ku/ligaseIV independent



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Non Homologous End JoiningNon Homologous End Joining• Ku 70/80 (or 86) heterocomplex tethers DNA and recruits DNA-PKcs that Ku 70/80 (or 86) heterocomplex tethers DNA and recruits DNA-PKcs that

promotes binding of various proteins: promotes binding of various proteins: • NucleasesNucleases that remove damaged DNA that remove damaged DNA

– Artemis/DNA-PKcs bind to form a 5Artemis/DNA-PKcs bind to form a 5’’ to 3 to 3’’ endonuclease that makes blunt endonuclease that makes blunt ends ends

– DNA-PK is activated on binding DNADNA-PK is activated on binding DNA– Autophosphorylation aids binding of other repair proteins Autophosphorylation aids binding of other repair proteins

• PolymerasesPolymerases that lay down the nucleotide structure that lay down the nucleotide structure– Pol X family members Pol X family members and and and TdT that have varying degrees of and TdT that have varying degrees of

template dependency. template dependency. pol can add nucleotides randomly to generate pol can add nucleotides randomly to generate microhomology that assists repairmicrohomology that assists repair

• LigasesLigases restore the phosphodiester backbone restore the phosphodiester backbone– Cernunnos (XLF)/XRCC4/DNA ligase IV complexCernunnos (XLF)/XRCC4/DNA ligase IV complex– XRCC4/DNA ligase IV are flexible being able to ligate just one strand or XRCC4/DNA ligase IV are flexible being able to ligate just one strand or

across gapsacross gaps

• Each enzyme has a range of flexibilities, allowing many outcomesEach enzyme has a range of flexibilities, allowing many outcomes


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V1V1 J3J3J2J2J1J1V29V29V3V3V2V2 J4J4C3C3C2C2 C4C4C1C1

Ig L chainIg L chain

Stem cellStem cell

V1V1 V3V3V2V2 J3J3 J4J4C3C3 C4C4

J2J2J1J1

V29V29C2C2

C1C1

VDJ rejoining in Ab FormationVDJ rejoining in Ab Formation

RAG 1 and RAG 2 endonucleases RAG 1 and RAG 2 endonucleases make DSB that are re-annealed bymake DSB that are re-annealed byNHEJ to make functional Ig or TCR.NHEJ to make functional Ig or TCR.

B cellB cell


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NHEJ apparatusNHEJ apparatus

XRCC4XRCC4LIGASE IVLIGASE IV

KU 70/80 heterodimer recruits DNA-PKcs, its kinase is activated on binding to DNA 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. and it autophosphorylates to bind Artemis that processes overhangs to blunt ends. The Cernunnos/DNA-ligase IV/XRCC4 complex then ligates the DNA.The Cernunnos/DNA-ligase IV/XRCC4 complex then ligates the DNA.

CernunnosCernunnos

ArtemisArtemis

DNA-Protein Kinase (DNA-Pk) DNA-Protein Kinase (DNA-Pk) phosphorylatesphosphorylates

P53, c-jun - apoptosis, etc.P53, c-jun - apoptosis, etc.eIF-2 - inhibition of protein synthesiseIF-2 - inhibition of protein synthesisH2AX - histone phosphorylationH2AX - histone phosphorylation

KUKU70/8070/80

KUKU70/8070/80

DNA-PKcsDNA-PKcs(catalytic subunit)(catalytic subunit)

PPPPPPPP

PPPPPPPP

PPp


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DNA-PKDNA-PK

• Only protein known to be activated by binding DSBOnly protein known to be activated by binding DSB• Required for DNA DSB repair and V(D)J rejoining by Required for DNA DSB repair and V(D)J rejoining by

NHEJNHEJ• Composed of DNA-PKcs (p450), KU70, Ku80Composed of DNA-PKcs (p450), KU70, Ku80• A large molecule - 4127 aa, 470kDa, 180 kbpA large molecule - 4127 aa, 470kDa, 180 kbp• Is a ser/thr kinase with homology to PI-3 kinase, but has Is a ser/thr kinase with homology to PI-3 kinase, but has

no lipid activity. no lipid activity. • Scid mice defective in DNA-PKcsScid mice defective in DNA-PKcs• Most Scid humans are defective in Artemis, which is Most Scid humans are defective in Artemis, which is

phosphorylated by DNA-PK and binds to DNA DSB to phosphorylated by DNA-PK and binds to DNA DSB to form an endonucleaseform an endonuclease

Foci are formed that act as an amplification platform


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0Gy0Gy

1Gy1Gy2hr2hr

1Gy1Gy24hr24hr

Cont XRCC3-ve DNA-PKcs-veCont XRCC3-ve DNA-PKcs-ve

-H2AX foci -H2AX foci function to function to

stabilize DSB stabilize DSB In DNA-PKcs In DNA-PKcs cells, they are cells, they are

more numerous more numerous after irradiation after irradiation and persist for and persist for

24hrs24hrs


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The The Mre11/ Rad50/ NBS1 (MRN) ComplexMre11/ Rad50/ NBS1 (MRN) Complex is involved as a tether for is involved as a tether forDSB for HRDSB for HRMre11 has nuclease activityMre11 has nuclease activity- NBS = Nijmegen Breakage Syndrome protein (nibrin) binds ATMNBS = Nijmegen Breakage Syndrome protein (nibrin) binds ATM- Nijmegen Breakage Syndrome patients areNijmegen Breakage Syndrome patients are

- Radiation sensitiveRadiation sensitive- Have microcephalyHave microcephaly- Immune deficienciesImmune deficiencies- Predisposition to lymphoid malignanciesPredisposition to lymphoid malignancies- Cells showCells show

- defect in DSB repairdefect in DSB repaircell cycle arrest abnormalitiescell cycle arrest abnormalities

- Including radio-resistant DNA synthesisIncluding radio-resistant DNA synthesis


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Homologous Homologous RecombinationRecombination

mis-match repair ofheteroduplex DNA

++ ++ ++

dsbDNA polymerase

blocked

Strand invasion

resolutionof Hollidayjunction

single strand gap fill

Involved in stalled replication forks as well as DSB repair Involved in stalled replication forks as well as DSB repair Several complex mechanisms involvedSeveral complex mechanisms involved

MRN

Rad51BRCA2§

Rad 50ATM

MDC1

Rad52

5’ to 3’ resection

DNA polymerases and ligases

-H2AX -H2AX


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• Chromatin structure decondenses at site of Chromatin structure decondenses at site of DSBDSB

• Histone acetylation and ubiquitination Histone acetylation and ubiquitination involved in DNA repairinvolved in DNA repair


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DNA-PKDNA-PK ATRATRBRCA1 BRCA1

Rad51/52Rad51/52

HRHRNHEJNHEJ

DNA DSBsDNA DSBs

SIGNAL TRANSDUCTIONSIGNAL TRANSDUCTION

DNA DAMAGE RESPONSEDNA DAMAGE RESPONSE

MRE11, Rad50, Nbs1MRE11, Rad50, Nbs1Ku 70/80Ku 70/80SensorsSensors

KinasesKinases

Cell Cycle Arrest, Apoptosis, DNA repairCell Cycle Arrest, Apoptosis, DNA repair

Effector Effector proteinsproteins

Relay Relay proteinsproteins

DNA DSB repair activates signaling with cellular consequences!

ATMATM


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DNA DAMAGE RESPONSEDNA DAMAGE RESPONSE

ATMATM ATRATR

DNA DSBsDNA DSBs

p53p53mdm2mdm2

P*P*

p21p21 BaxBax

G1/S arrestG1/S arrest apoptosisapoptosis

CHK2CHK2

MRNMRN

DNA repairDNA repair

CHK1CHK1

G2 arrest G2 arrest S phase delayS phase delay

UV damage,UV damage,Cross-linking agentsCross-linking agents

p53 degradationp53 degradation

BRCA1/2BRCA1/2Rad51Rad51

FocusFocusformationformation

DNA-PkDNA-PkH2AXH2AX

CDC25CDC25

KinaseKinase

PhosphatasePhosphatase

HRNHEJ


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• DNA repair genes are genomic DNA repair genes are genomic ““caretakercaretaker”” genes genes preventing cancer by removing DNA mutationspreventing cancer by removing DNA mutations

• Defects in DNA repair genes are very common in Defects in DNA repair genes are very common in cancerscancers

• Loss of many DNA repair genes is embryonic lethal Loss of many DNA repair genes is embryonic lethal or results in genomic instabilityor results in genomic instability

• Individuals who are Individuals who are ‘‘carrierscarriers’’ of defective DNA repair of defective DNA repair genes may be especially sensitive to irradiation and genes may be especially sensitive to irradiation and radiation-induced cancers and may be 5-10% of the radiation-induced cancers and may be 5-10% of the populationpopulation– Epidemiological studies have shown that AT Epidemiological studies have shown that AT

heterozygotes have a predisposition for cancer, heterozygotes have a predisposition for cancer, especially for breast cancer in women. especially for breast cancer in women.


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Autosomal Recessive Disorders with Repair DefectsAutosomal Recessive Disorders with Repair Defects• Xeroderma pigmentosum (XP) and related CockayneXeroderma pigmentosum (XP) and related Cockayne’’s syndrome s syndrome

–U.V. sensitivityU.V. sensitivity–At least 7 genes (ERCC 1-6; excision repair cross complementing)At least 7 genes (ERCC 1-6; excision repair cross complementing)–DNA binding and damage recognition, helicase, endonucleases, transcription DNA binding and damage recognition, helicase, endonucleases, transcription factors, inability to excise dimersfactors, inability to excise dimers

• FanconiFanconi’’s anemias anemia–Mutated in 90% aplastic anemias, commonly in leukemias, 20% solid tumors Mutated in 90% aplastic anemias, commonly in leukemias, 20% solid tumors –Sensitivity to X-linking agents (e.g. mitomycin C) - genomic instabilitySensitivity to X-linking agents (e.g. mitomycin C) - genomic instability–7 genes cloned (A, C, D1, D2, E, F, G); D1 is BRCA27 genes cloned (A, C, D1, D2, E, F, G); D1 is BRCA2

• BloomBloom’’s and Werners and Werner’’s syndromess syndromes–Helicases mutatedHelicases mutated–Defective recombination and replicationDefective recombination and replication–Cancer predispositionCancer predisposition

• Li Fraumeni syndromeLi Fraumeni syndrome–Rare autosomal dominantRare autosomal dominant–Breast, soft tissue, bone sarcomas with multiple primaries in childhoodBreast, soft tissue, bone sarcomas with multiple primaries in childhood–70% have p53 mutations, others have CHK2 mutations70% have p53 mutations, others have CHK2 mutations

• Ataxia telangiectasiaAtaxia telangiectasia• Nijmegen-breakage syndromeNijmegen-breakage syndrome


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Ataxia TelengiectasiaAtaxia Telengiectasia• Rare autosomal recessive - 1:20,000-1:1000,000, described in 1920s Rare autosomal recessive - 1:20,000-1:1000,000, described in 1920s • 70-250 fold excess of leukemia/lymphoma and carcinomas (1960s) 70-250 fold excess of leukemia/lymphoma and carcinomas (1960s) • Sensitive to ionizing radiation (1974)Sensitive to ionizing radiation (1974)• Cerebellar degeneration, progressive ataxia, telangiectasia, immune Cerebellar degeneration, progressive ataxia, telangiectasia, immune

deficiency (T and B)deficiency (T and B)• Chromosomal instability, DSB repair defect, initial damage unalteredChromosomal instability, DSB repair defect, initial damage unaltered• Signal transduction defect - low, late p53/ GADD45/ c-jun inductionSignal transduction defect - low, late p53/ GADD45/ c-jun induction• No G1 arrest, no S phase delay (RDS), G2 arrest alteredNo G1 arrest, no S phase delay (RDS), G2 arrest altered• AT gene (1995) homology to phosphoinositol-3 kinase superfamilyAT gene (1995) homology to phosphoinositol-3 kinase superfamily• ATM truncations and missense might give different outcomesATM truncations and missense might give different outcomes• Missense found in 8% of breast cancer patients, 20%CLL. No increase in Missense found in 8% of breast cancer patients, 20%CLL. No increase in

truncations.truncations.• AT heterozygotes have 1.3-2.9 fold increase in breast cancer risk. No AT heterozygotes have 1.3-2.9 fold increase in breast cancer risk. No

obvious increase in cytotoxic radiosensitivity.obvious increase in cytotoxic radiosensitivity.


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Nijmegen Breakage SyndromeNijmegen Breakage Syndrome

• Nibrin (Nbs) gene on chromosome 8q21Nibrin (Nbs) gene on chromosome 8q21• Microcephaly, growth and mental retardationMicrocephaly, growth and mental retardation• High leukemia riskHigh leukemia risk• Radiation sensitivityRadiation sensitivity• Late, low p53, lack G1/S arrestLate, low p53, lack G1/S arrest• Nibrin binds in MRE11, RAD50 (MRN) complexNibrin binds in MRE11, RAD50 (MRN) complex

• AT-Like Disorder (ATLD)AT-Like Disorder (ATLD) is an Mre11 defectis an Mre11 defect


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BRCA1 and BRCA2 Tumor Suppressor GenesBRCA1 and BRCA2 Tumor Suppressor Genes

• BRCA 1:BRCA 1:– Average 65 % lifetime risk for breast cancerAverage 65 % lifetime risk for breast cancer– 40 percent to 60 percent lifetime risk for second breast cancer (not 40 percent to 60 percent lifetime risk for second breast cancer (not

reappearance of first tumor)reappearance of first tumor)– Average 39 percent lifetime risk for ovarian cancerAverage 39 percent lifetime risk for ovarian cancer– Increased risk for other cancer types, such as prostate cancerIncreased risk for other cancer types, such as prostate cancer– BRCA1 cancers tend to be BRCA1 cancers tend to be ““basal-likebasal-like””, ER-ve, ER-ve– Expressed in proliferating cells at G1/SExpressed in proliferating cells at G1/S– Associate with rad51 which is involved in DSB repair in HRAssociate with rad51 which is involved in DSB repair in HR

• BRCA2 is FANC-D1BRCA2 is FANC-D1– Average 45 % lifetime risk for breast cancer in females, 6% in malesAverage 45 % lifetime risk for breast cancer in females, 6% in males– Average 11 percent lifetime risk for ovarian cancerAverage 11 percent lifetime risk for ovarian cancer– Increased risk for other cancer types, such as pancreatic, prostate, Increased risk for other cancer types, such as pancreatic, prostate,

laryngeal, stomach cancer, and melanomalaryngeal, stomach cancer, and melanoma

• Cells with mutated BRCA 1/2 are slightly more sensitive to radiation, Cells with mutated BRCA 1/2 are slightly more sensitive to radiation, cisplatin and MMC because of their role in homologous recombinationcisplatin and MMC because of their role in homologous recombination


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Human Chromosome Instability Human Chromosome Instability and Radiosensitivityand Radiosensitivity

SyndromeSyndrome GeneGene DefectDefect XIRXIR

ATAT

NBSNBS

ATLDATLD

Li-FraumeniLi-Fraumeni

FanconiFanconi’’s Anemias AnemiaFamilial Breast CaFamilial Breast Ca

BloomBloom’’ss

WernerWerner’’ss

Lig4Lig4

SCIDSCID

ATMATM

NBS1NBS1

MRE11MRE11

P53/CHK2P53/CHK2

FANA-GFANA-G

BRCA1/2BRCA1/2

BLM helicaseBLM helicase

WRN helicaseWRN helicase

Ligase4Ligase4

ArtemisArtemis

SensorSensor

Sensor?Sensor?

Sensor?Sensor?

Sensor?Sensor?

HRHR

HRHR

ReplicationReplication

ReplicationReplication

NHEJNHEJ

NHEJNHEJ

++++++++

++++

++++

+/-+/-

+/-+/-

++

--

--

++++

++++


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Questions onQuestions onRadiation Targets 1: Radiation Targets 1:

DNA, Chromosome and Chromatid Damage and RepairDNA, Chromosome and Chromatid Damage and Repair


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What is the most common form of DNA What is the most common form of DNA damage existing in cells under normal damage existing in cells under normal conditions?conditions?

1.1. Double strand breaksDouble strand breaks

2.2. Apurinic/apyrimidinic sitesApurinic/apyrimidinic sites

3.3. Interstrand crosslinksInterstrand crosslinks

4.4. Thymidine dimer formationThymidine dimer formation


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What assay would be the most sensitive to measure What assay would be the most sensitive to measure radiation-induced DNA double strand breaks?radiation-induced DNA double strand breaks?

1.1. Neutral elution of DNANeutral elution of DNA

2.2. H2AX focus formationH2AX focus formation

3.3. Comet assay under neutral conditionsComet assay under neutral conditions

4.4. Pulsed field electrophoresis of DNA Pulsed field electrophoresis of DNA


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What radiation damage is measured by the alkaline What radiation damage is measured by the alkaline elution of DNA techniqueelution of DNA technique

1.1. Single strand breaksSingle strand breaks

2.2. Base damageBase damage

3.3. Double strand breaksDouble strand breaks

4.4. Single and double strand breaksSingle and double strand breaks

5.5. DNA-protein crosslinksDNA-protein crosslinks


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Which of the following is true for the Which of the following is true for the H2AX focus H2AX focus formation assay?formation assay?

1.1. It measures the ability of radiation to transform It measures the ability of radiation to transform normal cells towards cancernormal cells towards cancer

2.2. Most foci are seen at 24 hoursMost foci are seen at 24 hours

3.3. The foci that form after about 10mins The foci that form after about 10mins approximate to the number of radiation-induced approximate to the number of radiation-induced DNA DSBsDNA DSBs

4.4. The foci are dependent of activation of ATM The foci are dependent of activation of ATM kinasekinase


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The micronucleus assayThe micronucleus assay1.1. Is a measure of DNA damageIs a measure of DNA damage2.2. Measures fragments of nuclei that are lost at Measures fragments of nuclei that are lost at

mitosismitosis3.3. Is a measure of histone damageIs a measure of histone damage4.4. Measure micronuclei formed by chromosome Measure micronuclei formed by chromosome

translocationstranslocations5.5. Uses microRNA techniques to measure DNA Uses microRNA techniques to measure DNA

damagedamage


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Radiation-induced chromosome damage Radiation-induced chromosome damage that is usually lethal is most likely due tothat is usually lethal is most likely due to

1.1. DeletionsDeletions

2.2. TranslocationTranslocation

3.3. Exchange-type aberrationsExchange-type aberrations

4.4. Gene lossGene loss

5.5. Dicentics or ringsDicentics or rings


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Which of the following is correct about sub-Which of the following is correct about sub-lethal damage repair. It occurs lethal damage repair. It occurs

1.1. When cells are held in a non-proliferating When cells are held in a non-proliferating statestate

2.2. Between fractions of radiationBetween fractions of radiation

3.3. At the G1/s checkpointAt the G1/s checkpoint

4.4. Only at low radiation dosesOnly at low radiation doses


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Which of the following repair mechanisms is Which of the following repair mechanisms is most important after X-ray exposure of cellsmost important after X-ray exposure of cells

1.1. Mismatch repairMismatch repair

2.2. Nucleotide excision repairNucleotide excision repair

3.3. Double strand break repairDouble strand break repair

4.4. Base excision repairBase excision repair


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Double strand breaks are least likely to Double strand breaks are least likely to contribute to DNA lesions in which of the contribute to DNA lesions in which of the following situationsfollowing situations1.1. Cellular respirationCellular respiration2.2. VDJ rejoining to make antibodiesVDJ rejoining to make antibodies3.3. MeiosisMeiosis4.4. Antibody class switchingAntibody class switching


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What sets DNA repair of double strand breaks by What sets DNA repair of double strand breaks by homologous recombination apart from non homologous recombination apart from non homologous end joining mechanisms? Its homologous end joining mechanisms? Its involvement ininvolvement in

1.1. G1 cell cycle phase onlyG1 cell cycle phase only

2.2. Cell cycle phases other than G1Cell cycle phases other than G1

3.3. All cell cycle phasesAll cell cycle phases

4.4. Increasing genomic instabilityIncreasing genomic instability


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What sets DNA repair of double strand breaks by What sets DNA repair of double strand breaks by homologous recombination apart from non homologous recombination apart from non homologous end joining mechanismshomologous end joining mechanisms

1.1. It does not involve the MRN complexIt does not involve the MRN complex

2.2. It involves ligasesIt involves ligases

3.3. It activates the BRCA tumor suppressor proteinIt activates the BRCA tumor suppressor protein

4.4. It is error-proneIt is error-prone


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Which of the following is NOT true concerning DNA Which of the following is NOT true concerning DNA protein kinase? It is protein kinase? It is 1.1. Critical for DNA DSB repair via the Critical for DNA DSB repair via the

nonhomologous end joining pathwaynonhomologous end joining pathway2.2. Formed from Ku proteins and DNA-PK catalytic Formed from Ku proteins and DNA-PK catalytic

subunitsubunit3.3. Activated on binding DNA DSBActivated on binding DNA DSB4.4. Defective in many humans with severe combined Defective in many humans with severe combined

immune deficiency (Scid) diseaseimmune deficiency (Scid) disease5.5. Defective in scid miceDefective in scid mice


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Which of the following is true about DNA repair Which of the following is true about DNA repair genesgenes

1.1. They are They are ““gatekeepergatekeeper”” genes that directly genes that directly regulate tumor growth by inhibiting growth or by regulate tumor growth by inhibiting growth or by promoting cell deathpromoting cell death

2.2. They are They are ““caretakercaretaker”” genes that prevent cancer- genes that prevent cancer-causing mutationscausing mutations

3.3. There are about 20 in human cellsThere are about 20 in human cells

4.4. Loss of an individual gene is not commonly a Loss of an individual gene is not commonly a problem as the system is highly redundant problem as the system is highly redundant


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Which of the following in NOT true about Ataxia Which of the following in NOT true about Ataxia Telengiectasia lymphoblastoid cellsTelengiectasia lymphoblastoid cells1.1. They have a D0 of about 50cGyThey have a D0 of about 50cGy2.2. They show a higher than normal level of initial They show a higher than normal level of initial

DNA double strand breaks after exposure to DNA double strand breaks after exposure to ionizing irradiationionizing irradiation

3.3. They show no G1 arrest, but normal S phase They show no G1 arrest, but normal S phase arrest after radiation exposurearrest after radiation exposure

4.4. They are slow to elevate p53 following radiation They are slow to elevate p53 following radiation exposureexposure


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Which of the following sets Nijmegan Breakage Which of the following sets Nijmegan Breakage Syndrome apart from Ataxia TelengiectasiaSyndrome apart from Ataxia Telengiectasia

1.1. Cells show less sensitivity to ionizing radiationCells show less sensitivity to ionizing radiation

2.2. Patients have no immune deficiencyPatients have no immune deficiency

3.3. Patients have no neurological problemsPatients have no neurological problems

4.4. Cells show normal cell cycle arrest following Cells show normal cell cycle arrest following irradiationirradiation

5.5. It is part of the MRN complexIt is part of the MRN complex


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AnswersAnswers

1. NA2. 23. 24. 45. 36. 27. 58. 29. 310. 111. 212. 313. 414. 315. 316. 5

objectives: know the limitations of different assays for different types of dna strand breaks

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