1. www.radbiol.ucla.edu WMcB2008 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

2. www.radbiol.ucla.edu WMcB2008 Objectives: Know the limitations of different assays for different types of DNA strand breaksKnow the limitations of different assays for different types of DNA strand breaks Know the different types of DNA and chromosome radiation damageKnow the different types of DNA and chromosome radiation damage Understand that multiple DNA repair mechanisms exist and whyUnderstand that multiple DNA repair mechanisms exist and why Be able to discuss repair of base, single strand and double strand DNA breaksBe 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 pathwaysKnow 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 pathwayUnderstand how DNA repair activates the DNA damage response pathway Recognize the role of DNA repair mutations in carcinogenesisRecognize the role of DNA repair mutations in carcinogenesis

3. www.radbiol.ucla.edu WMcB2008 DNA repair enzymes continuously monitor chromosomes to correct damaged nucleotidesDNA 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 damageApurinic/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 gapThe 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!Each time a cell divides it forms 10DSBs, and 50,000 SS!

4. www.radbiol.ucla.edu WMcB2008 Failure to repair leads to block in DNA replication, permanent cell cycle arrest, senescence, or deathFailure 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 carcinogenesisThese are the barriers that prevent development of genomic instability and carcinogenesis

5. www.radbiol.ucla.edu WMcB2008 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”

6. www.radbiol.ucla.edu WMcB2008 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, respectivelyNeutral 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 DSBsComet 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 assayDNA unwinding assay - a research assay Pulsed field gel electrophoresis - research assay that needs a high radiation dosePulsed field gel electrophoresis - research assay that needs a high radiation dose Quantification of damaged bases - a very specific assay, mainly for oxidative stressQuantification of damaged bases - a very specific assay, mainly for oxidative stress PCR-based assays - new range of research assays that still require validationPCR-based assays - new range of research assays that still require validation Chromosome/chromatid assays Micronucleus formation - classic assay especially in occupational exposureMicronucleus formation - classic assay especially in occupational exposure Chromosome/ chromatid aberration - classic exposure dosimetric assayChromosome/ chromatid aberration - classic exposure dosimetric assay –Conventional staining –Banding –FISH Assays

7. www.radbiol.ucla.edu WMcB2008 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

8. www.radbiol.ucla.edu WMcB2008 Fraction number % DNA retained 0Gy 5Gy 10Gy 20Gy 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 As # of breaks increase, the amount of DNA eluted through the filter increases Filter Assay

9. www.radbiol.ucla.edu WMcB2008 Comet Assay DOSEelectrophoresisagarose – + 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

10. www.radbiol.ucla.edu WMcB2008 H2AX is phosphorylated at the site of DNA DSBsH2AX is phosphorylated at the site of DNA DSBs Antibody to phosphorylated  H2AX reveals foci, the number of which approximates to the number of DSB.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.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 t 1/2 of about 2 hr  H2AX foci are apparent within a minute; reach a max in 10 min. Dephosphorylation starts after 30 minutes with a t 1/2 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 Formation 0Gy 1Gy 2hr 1Gy 24hr

11. www.radbiol.ucla.edu WMcB2008 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.

12. www.radbiol.ucla.edu WMcB2008 Pulsed-field gel electrophoresis – / + Molecular cut-off @ 10 Mbp Irradiate cells (10 Gy) and isolate DNA Irradiate cells (10 Gy) and isolate DNA Load in gel wellLoad in gel well Run gel with alternating pulses to force larger pieces of DNA into the gelRun gel with alternating pulses to force larger pieces of DNA into the gel Measure amount of DNA migrating into the gel by fluorescence or radiolabelMeasure amount of DNA migrating into the gel by fluorescence or radiolabel As the # of breaks increase, the amount of DNA migrating into gel increasesAs the # of breaks increase, the amount of DNA migrating into gel increases

13. www.radbiol.ucla.edu WMcB2008 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.

14. www.radbiol.ucla.edu WMcB2008 Micronucleus assay Typically, cells are cultured cells with cytochalasin B to induce metaphase arrest and then stained for DNA. 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.

15. www.radbiol.ucla.edu WMcB2008 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 G 1 irradiationG 1 irradiation Both sister chromatids involvedBoth sister chromatids involved Chromatid aberrations S or G 2 irradiationS or G 2 irradiation Usually only 1 chromatid involvedUsually only 1 chromatid involved Chromosome/Chromatid Aberrations There are 2 basic types of lesion Deletion-typeDeletion-type Exchange-typeExchange-type Mitosis G1G1 G2G2 S phase

16. www.radbiol.ucla.edu WMcB2008 Deletions DNA stain May be stable or unstable Fragments are lost at mitosis and may form micronuclei

17. www.radbiol.ucla.edu WMcB2008 Exchange-Type Rearrangements From: Hall “Radiobiology for the Radiologist” Are of two types: Symmetrical (balanced) Symmetrical (balanced) gene rearrangements - Generally stable - Translocations/Inversions Asymmetrical (not balanced) Asymmetrical (not balanced) - Generally lethal - Dicentrics / Rings fail at mitosis fail at mitosis cell death cell death

18. www.radbiol.ucla.edu WMcB2008 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 RingsDicentrics 1 2 3 4

19. www.radbiol.ucla.edu WMcB2008 Single break Sister union Inter-arm intra-change Inter-change 1 2 3 4 terminal deletion deletion translocation dicentric deletion & ring & ring & deletion ChromatidAberrations 1 2 3 4

20. www.radbiol.ucla.edu WMcB2008 Conventional Banding FISH fluorescence in situ hybridization CHROMOSOME ANALYSES

21. www.radbiol.ucla.edu WMcB2008 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

22. www.radbiol.ucla.edu WMcB2008 Multi-Color FISH in Human Lymphocyte Chromosomes Non-irradiatedIrradiated 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.

23. www.radbiol.ucla.edu WMcB2008 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.

24. www.radbiol.ucla.edu WMcB2008 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

25. www.radbiol.ucla.edu WMcB2008 Yield of radiation-induced chromosome damage Exchange-type “lethal” aberrations ≥ 2 hits requiredor 1 hit required P (2 hits) = D x D = D 2 Y (yield) = k + D 2 Y = k +  D 2 Y = k +  D 2 P( 1 hit) = D Y =  D Y =  D +  D 2

26. www.radbiol.ucla.edu WMcB2008 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 +  D2)

27. www.radbiol.ucla.edu WMcB2008 DNA Repair Classically, there are 2 typesClassically, there are 2 types Sub-Lethal and Potentially Lethal DamageSub-Lethal and Potentially Lethal Damage These are operationally-defined terms that differ in the the experimental set up in which they are demonstratedThese 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 clearThe molecular mechanisms may be similar, but this is not clear

28. www.radbiol.ucla.edu WMcB2008 Potentially Lethal Damage Potentially lethal damage is defined as damage that could cause death, but is modified by post-irradiation conditionsPotentially lethal damage is defined as damage that could cause death, but is modified by post-irradiation conditions

29. www.radbiol.ucla.edu WMcB2008 IRRADIATE At about 14 days count colonies calculate surviving fraction confluent cells trypsinize and plate at 0 min at 0 mintrypsinize and plate at 15 min at 15 mintrypsinize and plate at 30 min at 30 min Etc. time (mins) S.F. Potentially Lethal Damage Repair

30. www.radbiol.ucla.edu WMcB2008 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

31. www.radbiol.ucla.edu WMcB2008 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 qTo 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 + q  D2)S.F. = e –(  D + q  D2)

32. www.radbiol.ucla.edu WMcB2008 Redistribution Repair Repopulation 700R1500R Sublethal Damage Repair Assessed by varying the time between 2 or more doses of radiationAssessed by varying the time between 2 or more doses of radiation –Sometimes called Elkind-type repair

33. www.radbiol.ucla.edu WMcB2008 Some 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 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 RepairNucleotide 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 RepairDNA 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 RepairDouble Strand Break Repair

34. www.radbiol.ucla.edu WMcB2008 Enzymes exist that reverse rather than excise DNA damage existEnzymes exist that reverse rather than excise DNA damage exist –eg. MGMT (O 6 -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.”“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 Hegi et al NEJM 352:997-1003, 2005 The use of repair molecules and processes depends on a lot of factorsThe 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 commonThere 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

35. www.radbiol.ucla.edu WMcB2008 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

36. www.radbiol.ucla.edu WMcB2008 DSBs DSBs can be formed physiologically or pathologicallyDSBs can be formed physiologically or pathologically PhysiologicalPhysiological –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 PathologicalPathological –Ionizing radiation –ROS during cellular respiration –DNA replication across a nick –Enzymic action especially at fragile DNA sites –Topoisomerase failures

37. www.radbiol.ucla.edu WMcB2008 DSB Repair Recombination Models of DSB Repair #1 Homologous RecombinationHomologous 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/BRCA1/2MRN complex, Rad51/Rad52/XRCC2/ Rad54/BRCA1/2

38. www.radbiol.ucla.edu WMcB2008 Models of DSB Repair #2Models of DSB Repair #2 Non Homologous End Joining uses a non-homologous template with little or no microhomologyNon 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/ligaseIVKu70/Ku80 - Artemis/DNA-PKcs - Cerrunos/XRCC4/ligaseIV Microhomology-mediated end joining is an inefficient alternative that is Ku/ligaseIV independentMicrohomology-mediated end joining is an inefficient alternative that is Ku/ligaseIV independent DSB Repair

39. www.radbiol.ucla.edu WMcB2008 Non Homologous End Joining Ku 70/80 (or 86) heterocomplex tethers DNA and recruits DNA-PKcs that promotes binding of various proteins:Ku 70/80 (or 86) heterocomplex tethers DNA and recruits DNA-PKcs that promotes binding of various proteins: Nucleases that remove damaged DNANucleases 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 structurePolymerases 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 backboneLigases 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 outcomesEach enzyme has a range of flexibilities, allowing many outcomes

40. www.radbiol.ucla.edu WMcB2008 V1J3J2J1V29V3V2J4C3C2C4C1 Ig L chain Stem cell V1V3V2J3J4C3C4 J2J1 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

41. www.radbiol.ucla.edu WMcB2008 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 KU70/80 KU70/80 DNA-PKcs (catalytic subunit) PPPPPPPP PPpPPp

42. www.radbiol.ucla.edu WMcB2008 DNA-PK Only protein known to be activated by binding DSB Only protein known to be activated by binding DSB Required for DNA DSB repair and V(D)J rejoining by NHEJ Required for DNA DSB repair and V(D)J rejoining by NHEJ Composed of DNA-PKcs (p450), KU70, Ku80 Composed of DNA-PKcs (p450), KU70, Ku80 A large molecule - 4127 aa, 470kDa, 180 kbp A large molecule - 4127 aa, 470kDa, 180 kbp Is a ser/thr kinase with homology to PI-3 kinase, but has no lipid activity. Is a ser/thr kinase with homology to PI-3 kinase, but has no lipid activity. Scid mice defective in DNA-PKcs 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 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

43. www.radbiol.ucla.edu WMcB2008 0Gy1Gy2hr1Gy24hr Cont XRCC3-ve DNA-PKcs-ve  -H2AX foci function to stabilize DSB In DNA-PKcs cells, they are more numerous after irradiation and persist for 24hrs

44. www.radbiol.ucla.edu WMcB2008 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

45. www.radbiol.ucla.edu WMcB2008 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 MRNMRN Rad51 BRCA2 § Rad 50 ATM MDC1 Rad52 5’ to 3’ resection DNA polymerases and ligases  -H2AX

46. www.radbiol.ucla.edu WMcB2008 Chromatin structure decondenses at site of DSBChromatin structure decondenses at site of DSB Histone acetylation and ubiquitination involved in DNA repairHistone acetylation and ubiquitination involved in DNA repair

47. www.radbiol.ucla.edu WMcB2008 DNA-PKATR 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 Effectorproteins Relayproteins DNA DSB repair activates signaling with cellular consequences! ATM

48. www.radbiol.ucla.edu WMcB2008 DNA DAMAGE RESPONSE ATMATR DNA DSBs p53 mdm2 P* p21Bax G1/S arrest apoptosis CHK2 MRN DNA repair CHK1 G2 arrest S phase delay UV damage, Cross-linking agents p53 degradation BRCA1/2Rad51 Focusformation DNA-PkH2AX CDC25 Kinase Phosphatase HR NHEJ

49. www.radbiol.ucla.edu WMcB2008 DNA repair genes are genomic “caretaker” genes preventing cancer by removing DNA mutationsDNA repair genes are genomic “caretaker” genes preventing cancer by removing DNA mutations Defects in DNA repair genes are very common in cancersDefects in DNA repair genes are very common in cancers Loss of many DNA repair genes is embryonic lethal or results in genomic instabilityLoss 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 populationIndividuals 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.

50. www.radbiol.ucla.edu WMcB2008 Autosomal Recessive Disorders with Repair Defects Xeroderma pigmentosum (XP) and related Cockayne’s syndromeXeroderma 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 anemiaFanconi’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 syndromesBloom’s and Werner’s syndromes –Helicases mutated –Defective recombination and replication –Cancer predisposition Li Fraumeni syndromeLi Fraumeni syndrome –Rare autosomal dominant –Breast, soft tissue, bone sarcomas with multiple primaries in childhood –70% have p53 mutations, others have CHK2 mutations Ataxia telangiectasiaAtaxia telangiectasia Nijmegen-breakage syndromeNijmegen-breakage syndrome

51. www.radbiol.ucla.edu WMcB2008 Ataxia Telengiectasia Rare autosomal recessive - 1:20,000-1:1000,000, described in 1920sRare 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 deficiency (T and B)Cerebellar degeneration, progressive ataxia, telangiectasia, immune 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 truncations.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.AT heterozygotes have 1.3-2.9 fold increase in breast cancer risk. No obvious increase in cytotoxic radiosensitivity.

52. www.radbiol.ucla.edu WMcB2008 Nijmegen 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) is an Mre11 defectAT-Like Disorder (ATLD) is an Mre11 defect

53. www.radbiol.ucla.edu WMcB2008 BRCA1 and BRCA2 Tumor Suppressor Genes BRCA 1: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-D1BRCA2 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 recombinationCells with mutated BRCA 1/2 are slightly more sensitive to radiation, cisplatin and MMC because of their role in homologous recombination

54. www.radbiol.ucla.edu WMcB2008 Human Chromosome Instability and Radiosensitivity SyndromeGeneDefectXIR ATNBSATLDLi-Fraumeni Fanconi’s Anemia Familial Breast Ca Bloom’s Werner’s Lig4SCIDATMNBS1MRE11P53/CHK2FANA-GBRCA1/2 BLM helicase WRN helicase Ligase4ArtemisSensorSensor?Sensor?Sensor?HRHRReplicationReplicationNHEJNHEJ+++++++++/-+/-+--++++

55. www.radbiol.ucla.edu WMcB2008 Questions on Radiation Targets 1: DNA, Chromosome and Chromatid Damage and Repair

56. www.radbiol.ucla.edu WMcB2008 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

57. www.radbiol.ucla.edu WMcB2008 What assay would be the most sensitive to measure radiation-induced DNA double strand breaks? 1.Neutral elution of DNA  H2AX focus formation 3.Comet assay under neutral conditions 4.Pulsed field electrophoresis of DNA

58. www.radbiol.ucla.edu WMcB2008 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

59. www.radbiol.ucla.edu WMcB2008 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

60. www.radbiol.ucla.edu WMcB2008 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

61. www.radbiol.ucla.edu WMcB2008 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

62. www.radbiol.ucla.edu WMcB2008 Which of the following is correct about sub- lethal 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

63. www.radbiol.ucla.edu WMcB2008 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

64. www.radbiol.ucla.edu WMcB2008 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

65. www.radbiol.ucla.edu WMcB2008 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

66. www.radbiol.ucla.edu WMcB2008 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

67. www.radbiol.ucla.edu WMcB2008 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

68. www.radbiol.ucla.edu WMcB2008 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 cancer- causing 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

69. www.radbiol.ucla.edu WMcB2008 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

70. www.radbiol.ucla.edu WMcB2008 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

71. www.radbiol.ucla.edu WMcB2008 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