Objectives: Know the limitations of different assays for different types of DNA strand breaks

1. Radiation Targets 1: DNA, Chromosome and Chromatid Damage and RepairBill McBrideDept. Radiation OncologyDavid Geffen School MedicineUCLA, Los Angeles, Ca.wmcbride@mednet.ucla.edu

2. 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

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!

4. 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

5. Relevant Properties of DNA when Measuring Damage • 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 From: Watson et al. “Mol. Biol. of the Cell”

6. 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

7. Neutral and alkaline elution assays Alkaline/neutral conditions 2 days label cells with tritiated thymidine lyse cells 5% 10% 15% 20% spin sucrose gradient 0 Gy 5Gy 10Gy CPM Fraction # ALKALINE CONDITIONS UNWIND DNA AND MEASURES SSB and DSB NEUTRAL CONDITIONS MEASURES DSB

8. 0Gy 5Gy % DNA retained 10Gy 20Gy Fraction number Filter Assay Neutral elution (pH = 7.4) Alkaline elution for SSB+DSB (pH = 12.2) Irradiate cells Lyse cells on filter Vacuum elute Collect eluate and measure DNA concentration As # of breaks increase, the amount of DNA eluted through the filter increases

9. electrophoresis – + agarose Lysed cells – + 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

10. H2AX Focus Formation • H2AX is phosphorylated at the site of DNA DSBs • Antibody to phosphorylated H2AX reveals foci, the number of which approximates to the number of DSB. • DNA repair proteins are recruited to the same foci. • H2AX foci are apparent within a minute; reach a max in 10 min. Dephosphorylation starts after 30 minutes with a t1/2 of about 2 hr • The rate of repair and residual damage can be assessed within 24hrs 1Gy 2hr 1Gy 24hr 0Gy

11. TIME DNA Unwinding Assay Thisassay is based on the principle of alkaline unwinding of strandbreaks in double-stranded DNA to yield single-stranded DNA withthe number of strand breaks being proportional to the amountof DNA damage. Breaks are monitored by the fluorescence intensity of an intercalating dye, such as Hoechst 33258.

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

13. Irradiation 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.

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.

15. Mitosis G2 G1 S phase Chromosome/Chromatid Aberrations • Cytogenetic damage is normally assessed at first metaphase after irradiation. The type of cytogenetic damage depends upon where in the cell cycle the cell is when it is irradiated • Chromosome aberrations • G1 irradiation • Both sister chromatids involved • Chromatid aberrations • S or G2 irradiation • Usually only 1 chromatid involved • There are 2 basic types of lesion • Deletion-type • Exchange-type

16. Deletions May be stable or unstable Fragments are lost at mitosis and may form micronuclei DNA stain

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

18. 3 1 2 4 Chromosomal Aberrations IntrachromosomalInterchromosomal stable (non-lethal) Pericentric Inversions Translocations non-stable (lethal) Centric Rings Dicentrics 1 2 3 4 Single break Intra-arm intra-change Inter-arm intra-change Inter-change terminal interstitial paracentric pericentric deletion translocation dicentric deletion deletion inversion inversion & ring & deletion

19. 3 2 4 1 Chromatid Aberrations 1 2 3 4 Single break Sister union Inter-arm intra-change Inter-change terminal deletion deletion translocation dicentric deletion & ring & ring & deletion

20. CHROMOSOME ANALYSES Conventional Banding FISH fluorescence in situ hybridization

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

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.

23. 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. Yield of radiation-induced chromosome damage • Deletions • Terminal deletion = 1 hit • Chromatid deletion = 1 hit • Interstitial deletion = 2 hits • Yield (Y) ~ linear • Y = k +D • k = background  = proportionality Fate: Deletions lost at mitosis DOSE (Gy) Cornford and Bedford Rad Res 111: 385,1987

25. 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

26. 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)

27. 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

28. Potentially Lethal Damage • Potentially lethal damage is defined as damage that could cause death, but is modified by post-irradiation conditions

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

30. Sub-lethal (or accumulated) damage results from accumulation of events that individually are incapable of killing a cell but that together can be lethal 4 nm Repairable Sublethal Damage 2 nm Unrepairable Multiply Damaged Site

31. 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)

32. 700R 1500R Repopulation Redistribution Repair Sublethal Damage Repair • Assessed by varying the time between 2 or more doses of radiation • Sometimes called Elkind-type repair

33. 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

34. Enzymes exist that reverse rather than excise DNA damage exist • eg. MGMT (O6-methylguanine DNAmethyltransferase) removes methyl and other alkyl groups • “Patients with glioblastoma containing a methylatedMGMT promoter benefited from temozolomide, whereas those whodid not have a methylated MGMT promoter did not have such abenefit.” 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

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

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

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

38. DSB Repair • Models of DSB Repair #2 • Non Homologous End Joining uses a non-homologous template with little or no microhomology • Imprecise, makes mistakes (an advantage in the immune system) • Active at any time in cell cycle • Efficient at restoring chromosomal integrity • The major mechanism of DSB repair • Used physiologically in VDJ rejoining of T cell and Ig receptors • Mammalian mutants deficient in NHEJ are deficient in DNA repair and immunity (severe combined immune deficiency - scid - in mice and humans) • The major molecular players are: • Ku70/Ku80 - Artemis/DNA-PKcs - Cerrunos/XRCC4/ligaseIV • Microhomology-mediated end joining is an inefficient alternative that is Ku/ligaseIV independent

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

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

41. NHEJ apparatus LIGASE IV XRCC4 Artemis P P p Cernunnos PPPPPPPP KU 70/80 DNA-PKcs (catalytic subunit) KU 70/80 PPPPPPPP DNA-Protein Kinase (DNA-Pk) phosphorylates P53, c-jun - apoptosis, etc. eIF-2 - inhibition of protein synthesis H2AX - histone phosphorylation KU 70/80 heterodimer recruits DNA-PKcs, its kinase is activated on binding to DNA and it autophosphorylates to bind Artemis that processes overhangs to blunt ends. The Cernunnos/DNA-ligase IV/XRCC4 complex then ligates the DNA.

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

43. 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 0Gy 1Gy 2hr 1Gy 24hr

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

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

46. Chromatin structure decondenses at site of DSB • Histone acetylation and ubiquitination involved in DNA repair

47. DNA-PK DNA DAMAGE RESPONSE DNA DSBs NHEJ HR Sensors Ku 70/80 MRE11, Rad50, Nbs1 Rad51/52 Kinases BRCA1 ATM ATR Relay proteins SIGNAL TRANSDUCTION Effector proteins Cell Cycle Arrest, Apoptosis, DNA repair DNA DSB repair activates signaling with cellular consequences!

48. DNA DAMAGE RESPONSE UV damage, Cross-linking agents DNA DSBs DNA repair NHEJ HR H2AX DNA-Pk Focus formation MRN ATM ATR BRCA1/2 Rad51 CHK2 CHK1 Kinase P* mdm2 p53 Phosphatase CDC25 G2 arrest S phase delay p53 degradation p21 Bax G1/S arrest apoptosis

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.

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