The document summarizes various pathways for repairing DNA damage from radiation: base excision repair removes inappropriate bases; nucleotide excision repair removes bulky adducts like pyrimidine dimers. Mismatch repair fixes base-base mismatches. Non-homologous end joining and homologous recombination repair double-strand breaks, with the former being error-prone and active in G1, and the latter being error-free using a sister chromatid template and most active in G2 phase. Certain syndromes like ataxia-telangiectasia and LIG4 syndrome result from defects in these pathways and cause radiation sensitivity.

1. Repair of Radiation Damage & Dose Rate Effect How nucleotide excision repair protects against cancer Errol C. Friedberg Nature Reviews Cancer 1 , 22-33 (Oct 2001)

2. DNA Single Strand Repair

3. 3 Pathways for Excision of damaged or inappropriate bases Base Excision Repair Mismatch Repair Nucleotide Excision Repair

4. Base Excision Repair 1) U is 1 st removed by a glycosylase/DNA lyase 2) AP endonuclease then removes the sugar residue 3) Replacement w/correct nucleotide via DNA polymerase f3, 4) DNA ligase III-XRCC1-mediates ligation. * Most common types of DNA damage induced by ionizing radiation are repaired through base excision repair. U represents a putative single­base mutation

5. Base Excision Repair If >1 nucleotide mutated  RFC/PCNA/DNA polymerase 8/ E synthesizes the damaged strand  FENI endonuclease removes the Overhanging flap  Ligase I DNA strands are sealed

6. Nucleotide Excision Repair Nucleotide excision repair (NER) removes bulky adducts in the DNA, such as pyrimidine dimers

7. Nucleotide Excision Repair (1) Damage recognition (XPC/HHRAD23B (R23, XPA, RPA, TFIIH and XPG) -(2) Unwind (TFII), DNA incisions (XPG, ERCC1–XPF) around the lesion, usually 24 - 32 nucleotides in length -(3) Removal of the adducts -(4) Repair synthesis to fill in the gap (TFII (XPB, XPD) -(5) DNA ligation (DNA Ligase) How nucleotide excision repair protects against cancer Errol C. Friedberg Nature Reviews Cancer 1 , 22-33 (October 2001) e.g. Pyrimidine Dimer DNA Bubble

8. A mutation in NER genes does NOT lead to ionizing radiation sensitivity. Increases sensitivity to UV-induced DNA damage & alkylating agents that induce adducts. Nucleotide Excision Repair (NER) Germline mutations in NER genes lead to human DNA repair deficiency disorders such as xeroderma pigmentosum , in which patients are hypersensitive to UV light.

9. Mismatch Repair The mismatch repair pathway removes base-base mismatches in homologous recombination intermediates. Mutations in mismatch ( MSH, MLH, and PSM) repair genes lead to microsatellite instability Precise Mechanism unknown Microsatellite Instability (small base insertions or deletions) and cancer, especially hereditary nonpolyposis colon cancer (HNPCC). How nucleotide excision repair protects against cancer Errol C. Friedberg Nature Reviews Cancer 1 , 22-33 (October 2001)

10. DNA Double Strand Repair (including HRR and NHEJ) NHEJ is primary means of repairing dsDNA breaks Defects lead to: IR sensitivity, chromosome aberrations

11. DNA Double Strand Repair Non Homologous End- Joining (NHEJ) 1) Initiation (End recognition): - Binding of the Ku heterodimer to a dsDNA end 2) End Processing - Recruitment of DNA-dependent protein kinase catalytic subunit, DNAPKcs  Artemis activation - Artemis: Endonuclease dependent cleavage of overhangs 3) Synthesis : DNA polymerase 4) Bridging/Joining : XRCC4/DNA ligase IV/XLF complex DNAPK: functions as a regulatory component of NHEJ, potentially facilitating and regulating the processing of the DNA ends. Recruits complex of three proteins, XRCC4, DNA ligase IV and XLF, which carry out the final rejoining step. *Primary means of dsDNA break repair *Error prone* 2/2 in G1 w/no sister chromatid *Important for Ab diversity VDJ recombination

12. DNA Double Strand Repair Homologous Recombination (HR) ATM/ATR (damage sensor) PO4’s H2AX H2AX Recruits RAD51 RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52/BLM protects against exonucleolytic degradation. 4) Strand invasion (RAD54)  D-loop  Holliday jxn 5) Strand extension by DNA synthesis using sister chromatid template 6) Termination of the Holliday junction Template = Error-free *replication fork* -H2AX recruits BRCA2

14. NHEJ and HRR are not mutually exclusive, as both have been found to be active in the late S/G 2 phase of the cell cycle (but rare) DNA Double Strand Repair

15. No (uses template) Yes Sequence Loss Decreases No effect Proliferation Dependent Independent BRCA Dependent Independent ATM G2 G1 Phase of Cell Cycle No Yes Replication Associated HR NHEJ

16. DNA Repair Other Processes Single-strand annealing (SSA) plays a transitional role between HRR and NHEJ, and results in loss of genetic information owing to the exonuclease degradation of the DNA ends. Cross-Link repair pathways used for DNA-DNA or DNA-protein cross-link repair are still under in­vestigation.

17. PARP Nature Reviews Cancer 10 , 293-301 April 2010 OPINION:PARP inhibition: PARP1 and beyond Rouleau, Patel, Hendzel, Kaufmann & Poirier Synthetic Lethality = increases SSB w/in DNA large amounsts of SSBs collapse of replication forks during S phase Accumulation of DSBs *Central role in base excision repair so loss is Especially lethal in BRCA deficient cells as cant manage dsDNA Breaks

18. Syndromes that exhibit hypersentivity to ionizing radiation…

19. AT Cell-cycle checkpoints and cancer Michael B. Kastan and Jiri Bartek Nature 432 , 316-323(18 November 2004) doi:10.1038/nature03097

20. ATR

21. SCID SCID mice are immunodeficient due to defective DNA-PKcs Such cells are defective in nonhomologous recombination and are extremely radiosensitive. ·SCID humans are also immune deficient and radiosensitive owing to a mutation in Artemis . Cells defective in Artemis are defective in nonohomologous recombination and are radiosensitive .

22. SCID & NBS NBS (Nijmegen Breakage Syndrome) is a very rare disorder that results in increased cancer incidence. Cells defective in NBS lack an S phase checkpoint and are radiosensitive. ATLD (AT-Like Disorder) patients are clinically similar to AT patients except that their defect lies in the MRE7 7 gene . Cells from these patients are also sensitive to ionizing radiation.

23. Fanconi Anemia FA (Fanconi Anemia) patients are characterized by their hypersensitivity to cross-linking agents . Although fibroblasts derived from these patients are not sensitive to ionizing radiation , tumors arising in these patients are hypersensitive. The reasons for this are currently unknown.

24. Operational Classifications of Radiation Damage Radiation damage to mammalian cells can be divided into… Lethal damage – irreversible/irreparable and leads irrevocable to cell death Potentially lethal damage (PLD) – that which can be modified by postirradiation conditions Sublethal damage (SLD) – that which under normal conditions can be repaired unless additional sublethal damage is added

25. Potentially Lethal Damage The component of radiation damage that can be modified by manipulation of the postirradiation conditions is known as potentially lethal damage.

26. Potentially Lethal Damage Potentially lethal damage repair can occur if cells are prevented from dividing for 6 hours or more after irradiation ; this is manifest as an increase in survival. This repair can be demonstrated in vitro by keeping cells in saline or plateau phase for 6 hours after irradiation and in vivo by delayed removal and assay of animal tumors or cells of normal tissues.

27. Potentially Lethal Damage

28. Sublethal Damage The repair of sublethal damage is simply the repair of double-strand breaks . If a dose is split by a time interval, some double­strand breaks produced by the first dose are repaired before the second dose. Recall that double strand “lethal” damage may be formed by (1) single-track or (2) multiple-track damage. The component of cell killing resulting from single-track damage is the same whether the dose is a single exposure or fractionated. The same is not true of multiple-track damage.

29. Sublethal Damage

30. For x-rays, split fractionation separated by 1 to 4 hours, results in a marked increase in cell survival because of the prompt repair of sublethal damage (multi-track damage). For neutrons, split fractionation offers little repair of sublethal damage (single track damage). Sublethal Damage

31. Dose Rate Effect

32. Inverse Dose Rate

33. Dose Rate Effect There is a dose-rate effect, such that at very low dose rates (<0.01 G/min) there is minimal checkpoint activation, probably due to minimal ATM activation. At ~1 Gy/hr, only Late G2 checkpoint is triggered , leading to reassortment of cells at the G2/M interface (and resulting in the inverse dose rate effect). At higher dose rates , cell cycle progression is inhibited at all checkpoints EXAMPLE

34. Dose Rate Effect EXAMPLE

35. To better illustrate the concept of inverse dose rate consider that… At lower dose rates cells may continue to progress through the cell cycle until they are arrested in G 2 , the most radiosensitive phase of the cycle leading to greater cell kill. By contrast, at higher dose rates, cells may become &quot;frozen&quot; in a more radioresistant phase of the cycle leading to less cell kill. Inverse Dose Rate

36. Brachytherapy

37. Brachytherapy

38. Brachytherapy

39. Brachytherapy

40. Radiolabeled Ig Therapy

41. Radiolabeled Ig Therapy  

42. Radiolabeled Ig Therapy

43. Questions Which of the following is NOT a characteristic of DNA-protein kinase (DNA-PK)? A. DNA-PK consists of a catalytic subunit and two smaller accessory proteins, Ku70 (XRCC6) and Ku80 (XRCC5). B. It participates in the repair of DNA double strand breaks primarily through homologous recombination. C. Its loss in mice results in radiation sensitivity. D. DNA-PK phosphorylates histone H2AX at sites of double strand breaks. E. It is a phosphatidyl-inositol-3-kinase.

44. B DNA-PK is involved with non-homologous end-joining, not homologous recombination. Answer

45. Which of the following proteins is NOT involved in DNA repair? A. Artemis B. RAD51 C. DNA-PKcs D. CDK4 E. BRCA1 Questions

46. D CDK4 is a cyclin dependent kinase that plays an important role in the progression of cells through G 1 and into S phase. Artemis and DNA-PKcs play important roles in non-homologous end-joining of DNA double strand breaks, whereas RAD51 and BRCA1 are involved in the repair of double strand breaks through homologous recombination. Answer

47. G1/S Phase Checkpoint

48. SCID mice are often used in radiobiology research because they: A. are radioresistant B. exhibit high levels of non-homologous end joining C. have efficient immune systems D. are better able to repair radiation damage E. are useful hosts for growing human tumor xenografts Questions

49. E SCID mice are immune deficient, making them good hosts for growing xenografts of human tumors. SCID mice are deficient in DNA-PK and are therefore radiosensitive. Cells from these mice have low levels of non-homologous end-joining. Answer

50. Cells derived from individuals diagnosed with xeroderma pigmentosum are deficient in: A. nucleotide excision repair B. methyl-guanine transferase C. mismatch repair D. base excision repair Questions

51. A People with xeroderma pigmentosum are deficient in one of the several proteins involved in nucleotide excision repair . They are therefore extremely sensitive to UV irradiation because they are unable to repair the pyrimidine dimers produced in DNA, but they are not sensitive to ionizing radiation. Answer

52. A mutation in NER genes does NOT lead to ionizing radiation sensitivity. Increases sensitivity to UV-induced DNA damage & alkylating agents that induce adducts. Nucleotide Excision Repair (NER) Germline mutations in NER genes lead to human DNA repair deficiency disorders such as xeroderma pigmentosum, in which patients are hypersensitive to UV light.

53. Homologous recombinational repair of DNA double strand breaks is most likely to occur: A. in G 0 B. in G 1 C. in early S phase D. in G 2 E. throughout the cell cycle Questions

54. D Homologous recombinational repair requires the presence of a homologous DNA template, and is therefore most likely to occur following DNA replication in G2 phase (when complete, sister chromatids are present in the cell). Answer

56. Which syndrome is caused by a deficiency in the repair-associated protein MRE11? A. Werner’s syndrome B. Ataxia-Telangiectasia-like disorder C. Xeroderma Pigmentosum D. Bloom’s syndrome E. Cocayne’s syndrome Questions

57. B A deficiency in MRE11 results in an ataxia telangiectasia-like disorder. Answer

58. DNA Double Strand Repair Homologous Recombination (HR) ATM/ATR (damage sensor) PO4’s H2AX H2AX Recruits RAD51 RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52 protects against exonucleolytic degradation. 4) Strand invasion (RAD54)  D-loop  Holliday jxn 5) Strand extension by DNA synthesis using sister chromatid template 6) Termination of the Holliday junction Template = Error-free *replication fork* -H2AX recruits BRCA2

59. All the following statements are true concerning homologous recombinational repair of DNA double strand breaks, EXCEPT: A. H2AX phosphorylation represents an important step in the formation of repair foci. B. The BLM protein serves to coat single stranded DNA regions to prevent their degradation. C. The MRN complex assists in producing single stranded regions following the induction of a DNA double strand break. D. RAD51 is a recombinase and forms a nucleoprotein filament that facilitates strand invasion. E. ATM is activated following irradiation by auto-phosphorylation and conversion from an inactive dimer to the active monomer. Questions

60. B The RAD52/BLM protein is a helicase. RPA serves to coat single stranded DNA regions generated during homologous recombination to prevent their degradation. Answer

61. DNA Double Strand Repair Homologous Recombination (HR) ATM/ATR (damage sensor) PO4’s H2AX H2AX Recruits RAD51 RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52 protects against exonucleolytic degradation. 4) Strand invasion (RAD54)  D-loop  Holliday jxn 5) Strand extension by DNA synthesis using sister chromatid template 6) Termination of the Holliday junction Template = Error-free *replication fork* -H2AX recruits BRCA2

62. All of the following statements about non-homologous end joining (NHEJ) are true, EXCEPT: A. Artemis is primarily responsible for ligating broken DNA ends. B. DNA ligase IV forms a tight complex with XRCC4. C. DNA-PKcs associates with Ku70/80 to form the DNA-PK holo-enzyme. D. The Ku heterodimer has a high affinity for DNA ends and forms a close-fitting asymmetrical ring that threads onto a free end of DNA. E. NHEJ is an error-prone process. Questions

63. A The main role for Artemis is to cleave (through its nuclease activity) any residual DNA loops or hairpins that form during non-homologous end-joining. Answer

64. No (uses template) Yes Sequence Loss Decreases No effect Proliferation Dependent Independent BRCA Dependent Independent ATM G2 G1 Phase of Cell Cycle No Yes Replication Associated HR NHEJ

65. Which of the following is NOT a substrate for ATM? A. Ku70/80 (XRCC6/XRCC5) B. BRCA1 C. NBS1 D. p53 (TP53) E. CHK2 (CHEK2) Questions

66. A All of the proteins listed are substrates for ATM except for Ku70/80 . Ku70/80 involved in NHEJ NHEJ is ATM independent Answer

67. DNA Double Strand Repair Non Homologous End- Joining (NHEJ) 1) Initiation (End recognition): - Binding of the Ku heterodimer to a dsDNA end 2) End Processing - Recruitment of DNA-dependent protein kinase catalytic subunit, DNAPKcs  Artemis activation - Artemis: Endonuclease dependent cleavage of overhangs 3) Synthesis: DNA polymerase 4) Bridging/Joining: XRCC4/DNA ligase IV/XLF complex DNAPK: functions as a regulatory component of NHEJ, potentially facilitating and regulating the processing of the DNA ends. Recruits complex of three proteins, XRCC4, DNA ligase IV and XLF, which carry out the final rejoining step. *error prone* 2/2 in G1 w/no sister chromatid *Important for Ab diversity VDJ recombination

68. Which of the following statements is TRUE concerning DNA repair processes? A. Between 10-20% of the population is thought to be heterozygous for the types of mutations that are responsible for causing ataxia telangiectasia (AT). B. Non-homologous end-joining requires the involvement of a sister chromatid. C. Mutations in the genes that encode proteins involved in translesion DNA synthesis are typically present in people who develop hereditary non-polyposis colon cancer. D. The most common types of DNA damage induced by ionizing radiation are repaired through base excision repair. Questions

69. D The most common alterations produced in the DNA by radiation are base damages which are repaired by base excision repair , a repair process that is usually rapid and accurate. The proportion of the population that is heterozygous for the types of mutations that are found in people with AT – typically protein truncation mutations – is roughly 1-2%. Non-homologous end joining does not require a sister chromatid. Mutation of the genes involved with mismatch repair, primarily MSH2 and MLH1, are often present in people who develop hereditary non-polyposis colon cancer. Homologous recombination is a relatively error-free process. Answer

70. Normal tissue complications are most likely to be exhibited following conventional radiotherapy in patients suffering from: A. ataxia telangiectasia B. systemic lupus erythematosus C. Bloom's syndrome D. xeroderma pigmentosum E. Fanconi's anemia Questions

71. A Radiation injury would most likely occur in a person with ataxia telangiectasia . People with this syndrome are very sensitive to ionizing radiation due to the absence of functional ATM protein, which plays a central role in the repair of DNA double strand breaks and regulation of the cell cycle following irradiation. Answer

74. Repair of DNA double-strand breaks can be accomplished by which one of the following pathways? A. mismatch repair B. non-homologous end joining C. base excision repair D. nucleotide excision repair E. photoreactivation Questions

75. B Non-homologous end-joining represents the principal means by which human cells repair DNA double strand breaks . Mismatch repair is primarily responsible for correction of errors made during DNA replication. Base excision repair removes base damages. Nucleotide excision repair mainly removes bulky adducts from DNA such as UV-induced pyrimidine dimers and chemical adducts. Photoreactivation involves the action of DNA photolyase which is activated by long wavelength UV and visible light to split Answer

76. DNA Double Strand Repair (including HRR and NHEJ) Depiction of the two main processes for repairing DNA dsb in mammalian cells. In NHEJ, the break is processed and rejoined with the possibility of sequence information being lost and incorrect repair. With HR an intact homolog is used as a template to ensure correct error free repair. : Eur J Pharmacol. Author manuscript; available in PMC 2010 June 25. Eur J Pharmacol. 2009 December 25; 625(1-3): 151–155. Published online 2009 October 14. NHEJ is primary means of repairing dsDNA breaks Defects lead to: IR sensitivity, chromosome aberrations

77. RAD51 and BRCA2 function together: A. as inhibitors of cyclin dependent kinases. B. to phosphorylate H2AX and NBS1. C. to enhance apoptosis by inhibiting p53 (TP53). D. in the initial steps of homologous recombination. E. to play a central role in nucleotide excision repair. Questions

78. Answer D RAD51 and BRCA2 function DNA double strand breaks.

79. DNA Double Strand Repair Homologous Recombination (HR) ATM/ATR (damage sensor) PO4’s H2AX H2AX Recruits RAD51 RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52/BLM protects against exonucleolytic degradation. 4) Strand invasion (RAD54)  D-loop  Holliday jxn 5) Strand extension by DNA synthesis using sister chromatid template 6) Termination of the Holliday junction Template = Error-free *replication fork* -H2AX recruits BRCA2