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Radiation Targets 1: DNA Chromosome and Chromatid Damage and Repair PowerPoint Presentation

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Description : Check out this medical presentation on Ataxia-Telangiectasia, which is titled "Radiation Targets 1: DNA, Chromosome and Chromatid Damage and Repair", to know about the limitations of different assays for different types of DNA strand breaks, the different types of DNA and chromosome radiation damage, multiple DNA repair mechanisms, repair of base, single strand and double strand DNA breaks, the molecules involved in homologous recombination and non-homologous end joining and how these initiate DNA damage response pathways, DNA repair activates the DNA damage response pathway, and the role of DNA repair mutations in carcinogenesis.

Tags : Ataxia-Telangiectasia, DNA repair, mutation, Xeroderma pigmentosum, Fanconi’s anemia, Cockayne’s Syndrome

Published on : May 10, 2021 | Views : 2434 | Downloads : 0

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Slide 1-

Radiation Targets 1: DNA, Chromosome and Chromatid Damage and Repair Bill McBride Dept. Radiation Oncology David Geffen School Medicine UCLA, Los Angeles, Ca. wmcbride@mednet.ucla.edu
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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
Slide 3-

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 8-oxyguanines sites produced per day per cell under physiologic conditions 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 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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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 DNA when Measuring Damage From: Watson et al. “Mol. Biol. of the Cell”
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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 Assays
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sucrose gradient Neutral and alkaline elution assays label cells with tritiated thymidine 2 days lyse cells 5% 10% 15% 20% spin ALKALINE CONDITIONS UNWIND DNA AND MEASURES SSB and DSB NEUTRAL CONDITIONS MEASURES DSB CPM 0 Gy 5Gy 10Gy Fraction # Alkaline/neutral conditions
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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 Filter Assay
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Comet Assay DOSE – + 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 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 H2AX Focus Formation 0Gy 1Gy 2hr 1Gy 24hr
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DNA Unwinding Assay 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 – / + Molecular cut-off @ 10 Mbp 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 DNA migrating into gel increases
Slide 13-

PCR-Based assays Many qPCR-based assays have been described to measure DNA breaks and repair. Some introduce plasmids into cells, others examine in situ genes.
Slide 14-

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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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 Chromosome aberrations G1 irradiation Both sister chromatids involved Chromatid aberrations S or G2 irradiation Usually only 1 chromatid involved Chromosome/Chromatid Aberrations There are 2 basic types of lesion Deletion-type Exchange-type
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Deletions DNA stain May be stable or unstable Fragments are lost at mitosis and may form micronuclei
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Exchange-Type Rearrangements From: Hall “Radiobiology for the Radiologist” Are of two types: Symmetrical (balanced) gene rearrangements Generally stable Translocations/Inversions Asymmetrical (not balanced) Generally lethal Dicentrics / Rings fail at mitosis cell death
Slide 18-

Single break Intra-arm intra-change Inter-arm intra-change Inter-change 1 2 3 4 terminal interstitial paracentric pericentric deletion translocation dicentric deletion deletion inversion inversion & ring & deletion Chromosomal Aberrations Intrachromosomal Interchromosomal stable (non-lethal) Pericentric Inversions Translocations non-stable (lethal) Centric Rings Dicentrics
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Single break Sister union Inter-arm intra-change Inter-change 1 2 3 4 terminal deletion deletion translocation dicentric deletion & ring & ring & deletion Chromatid Aberrations
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Conventional Banding FISH fluorescence in situ hybridization CHROMOSOME ANALYSES
Slide 21-

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. normal abnormal
Slide 22-

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 DOSE (Gy) Deletions Terminal deletion = 1 hit Chromatid deletion = 1 hit Interstitial deletion = 2 hits Yield (Y) ~ linear Y = k +D k = background  = proportionality Fate: Deletions lost at mitosis Cornford and Bedford Rad Res 111: 385,1987
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Yield of radiation-induced chromosome damage Exchange-type “lethal” aberrations ≥ 2 hits required or 1 hit required P (2 hits) = D x D = D2 Y (yield) = k + D2 Y = k + bD2 P( 1 hit) = D Y = aD Y = aD + 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 –(aD + 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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IRRADIATE At about 14 days count colonies calculate surviving fraction confluent cells trypsinize and plate at 0 min trypsinize and plate at 15 min trypsinize and plate at 30 min Etc. time (mins) S.F. Potentially Lethal Damage Repair
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Repairable Sublethal Damage 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 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 –(aD + 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
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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 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 The use of repair molecules and processes depends on a lot of factors 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 Nucleases remove damaged DNA Polymerases lay down the new structures Ligases restore the phosphodiester backbone
Slide 35-

BER NER MMR Ligation Repair patch synthesis Ligation DNA N-glycosylases Recognize and remove damage AP lyase or endonuclease Cleave backbone DNA polymerase Fills gap Repair patch synthesis DNA ligase Rad14p Rad1p 5’, Rad2p 3’ incision Msh2/3 or Msh2/Msh6
Slide 36-

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
Slide 37-

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
Slide 38-

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 DSB Repair
Slide 39-

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
Slide 40-

V1 J3 J2 J1 V29 V3 V2 J4 C3 C2 C4 C1 Ig L chain Stem cell V1 V3 V2 J3 J4 C3 C4 J2 J1 V29 C2 C1 VDJ rejoining in Ab Formation RAG 1 and RAG 2 endonucleases make DSB that are re-annealed by NHEJ to make functional Ig or TCR. B cell
Slide 41-

NHEJ apparatus XRCC4 LIGASE IV 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. Cernunnos Artemis DNA-Protein Kinase (DNA-Pk) phosphorylates P53, c-jun - apoptosis, etc. eIF-2 - inhibition of protein synthesis H2AX - histone phosphorylation KU 70/80 KU 70/80 DNA-PKcs (catalytic subunit) PPPPPPPP PPPPPPPP P P p
Slide 42-

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
Slide 43-

0Gy 1Gy 2hr 1Gy 24hr Cont XRCC3-ve DNA-PKcs-ve g-H2AX foci function to stabilize DSB In DNA-PKcs cells, they are more numerous after irradiation and persist for 24hrs
Slide 44-

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
Slide 45-

Homologous Recombination mis-match repair of heteroduplex DNA + + + dsb DNA polymerase blocked Strand invasion resolution of Holliday junction single strand gap fill Involved in stalled replication forks as well as DSB repair Several complex mechanisms involved MRN Rad51 BRCA2 § Rad 50 ATM MDC1 Rad52 5’ to 3’ resection DNA polymerases and ligases g-H2AX
Slide 46-

Chromatin structure decondenses at site of DSB Histone acetylation and ubiquitination involved in DNA repair
Slide 47-

DNA-PK ATR BRCA1 Rad51/52 HR NHEJ DNA DSBs SIGNAL TRANSDUCTION DNA DAMAGE RESPONSE MRE11, Rad50, Nbs1 Ku 70/80 Sensors Kinases Cell Cycle Arrest, Apoptosis, DNA repair Effector proteins Relay proteins DNA DSB repair activates signaling with cellular consequences! ATM
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DNA DAMAGE RESPONSE ATM ATR DNA DSBs p53 mdm2 P* p21 Bax G1/S arrest apoptosis CHK2 MRN DNA repair CHK1 G2 arrest S phase delay UV damage, Cross-linking agents p53 degradation BRCA1/2 Rad51 Focus formation DNA-Pk H2AX CDC25 Kinase Phosphatase HR NHEJ
Slide 49-

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.
Slide 50-

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
Slide 51-

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.
Slide 52-

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
Slide 54-

Human Chromosome Instability and Radiosensitivity
Slide 55-

Questions on Radiation Targets 1: DNA, Chromosome and Chromatid Damage and Repair
Slide 56-

What is the most common form of DNA damage existing in cells under normal conditions? Double strand breaks Apurinic/apyrimidinic sites Interstrand crosslinks Thymidine dimer formation
Slide 57-

What assay would be the most sensitive to measure radiation-induced DNA double strand breaks? Neutral elution of DNA H2AX focus formation Comet assay under neutral conditions Pulsed field electrophoresis of DNA
Slide 58-

What radiation damage is measured by the alkaline elution of DNA technique Single strand breaks Base damage Double strand breaks Single and double strand breaks DNA-protein crosslinks
Slide 59-

Which of the following is true for the H2AX focus formation assay? It measures the ability of radiation to transform normal cells towards cancer Most foci are seen at 24 hours The foci that form after about 10mins approximate to the number of radiation-induced DNA DSBs The foci are dependent of activation of ATM kinase
Slide 60-

The micronucleus assay Is a measure of DNA damage Measures fragments of nuclei that are lost at mitosis Is a measure of histone damage Measure micronuclei formed by chromosome translocations Uses microRNA techniques to measure DNA damage
Slide 61-

Radiation-induced chromosome damage that is usually lethal is most likely due to Deletions Translocation Exchange-type aberrations Gene loss Dicentics or rings
Slide 62-

Which of the following is correct about sub-lethal damage repair. It occurs When cells are held in a non-proliferating state Between fractions of radiation At the G1/s checkpoint Only at low radiation doses
Slide 63-

Which of the following repair mechanisms is most important after X-ray exposure of cells Mismatch repair Nucleotide excision repair Double strand break repair Base excision repair
Slide 64-

Double strand breaks are least likely to contribute to DNA lesions in which of the following situations Cellular respiration VDJ rejoining to make antibodies Meiosis Antibody class switching
Slide 65-

What sets DNA repair of double strand breaks by homologous recombination apart from non homologous end joining mechanisms? Its involvement in G1 cell cycle phase only Cell cycle phases other than G1 All cell cycle phases Increasing genomic instability
Slide 66-

What sets DNA repair of double strand breaks by homologous recombination apart from non homologous end joining mechanisms It does not involve the MRN complex It involves ligases It activates the BRCA tumor suppressor protein It is error-prone
Slide 67-

Which of the following is NOT true concerning DNA protein kinase? It is Critical for DNA DSB repair via the nonhomologous end joining pathway Formed from Ku proteins and DNA-PK catalytic subunit Activated on binding DNA DSB Defective in many humans with severe combined immune deficiency (Scid) disease Defective in scid mice
Slide 68-

Which of the following is true about DNA repair genes They are “gatekeeper” genes that directly regulate tumor growth by inhibiting growth or by promoting cell death They are “caretaker” genes that prevent cancer-causing mutations There are about 20 in human cells Loss of an individual gene is not commonly a problem as the system is highly redundant
Slide 69-

Which of the following in NOT true about Ataxia Telengiectasia lymphoblastoid cells They have a D0 of about 50cGy They show a higher than normal level of initial DNA double strand breaks after exposure to ionizing irradiation They show no G1 arrest, but normal S phase arrest after radiation exposure They are slow to elevate p53 following radiation exposure
Slide 70-

Which of the following sets Nijmegan Breakage Syndrome apart from Ataxia Telengiectasia Cells show less sensitivity to ionizing radiation Patients have no immune deficiency Patients have no neurological problems Cells show normal cell cycle arrest following irradiation It is part of the MRN complex
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Answers 1. NA 2. 2 3. 2 4. 4 5. 3 6. 2 7. 5 8. 2 9. 3 10. 1 11. 2 12. 3 13. 4 14. 3 15. 3 16. 5