Dna response to radiotherapy

DNA RESPONSE TO RADIOTHERAPY

1. DNA RESPONSE TO RADIOTHERAPY DR SAILENDRA NARAYAN PARIDA SENIOR RESIDENT DEPARTMENT OF RADIOTHERAPY MAULANA AZAD MEDICAL COLLEGE

2. STRUCTURE OF DNA

3. MECHANISM OF DNA REPLICATION

4. TYPES OF IONIZING RADIATION Highly Ionizing Radiation 1. Neutrons 2. Alpha particles Sparsely Ionizing Radiation 1. X-rays 2. Gama rays

5. MECHANISM OF CELL DAMAGE

6. ENERGY DEPOSITION EVENTS Spurs - energy = 100eV 95 % of x-ray and gama rays energy deposition events are spurs. Blobs - energy = 100-500eV.Less frequent than spurs. Short tracks

7. DNA Damage Response: Sensors & Effectors of DNA damage

8. Types of dna damage

9. SSB and DSB A-normal DNA double helix B-break in single strand C-break in both strands but widely separated D-break in both strands directly opposite

10. TYPES OF DNA DAMAGE SINGLE STRAND BREAK Little biologic significance Repaired readily using opposite strand as template Defect may result in mutation More common 1000 SSBs per cell after 1-2Gy DOUBLE STRAND BREAK Most important lesions produced by radiation Defect in repair may result in cell killing,carcinogenesis or mutation Less common 40 DSBs per cell

11. HOW TO MEASURE DNA BREAKS Pulsed field gel electrophoresis(PFGE) Single cell electrophoresis(comet assay) DNA damage induced nuclear foci assay.

12. DNA Repair

13. DNA REPAIR MECHANISMS REPAIR OF SSBs Base excision repair Nucleotide excision repair REPAIR OF DSBs Homologous recombination repair Nonhomologous end joining Crosslink repair Mismatch repair

14. SSB DNA REPAIR MECHANISMS BASE EXCISION REPAIR It repairs the base damages. 2 types- -repair single nucleotide -repair multiple nucleotide BER defect may lead to an increased mutation rate but usually do not result in cellular radiosensitivity . Exception is mutation of XRCC1 gene.

15. BASE EXCISION REPAIR

16. SSB DNA REPAIR MECHANISMS BASE EXCISION REPAIR It repairs the base damages. 2 types- -repair single nucleotide -repair multiple nucleotide BER defect may lead to an increased mutation rate but usually do not result in cellular radiosensitivity . Exception is mutation of XRCC1 gene. NUCLEOTIDE EXCISION REPAIR Removes bulky adducts in the DNA such as pyrimidine dimers . 2 types- - global genome NER(both coding and noncoding regions) - transcription coupled NER(only actively transcribed areas) Defect leads to increased sensitivity to UV light and anticancer agents induced DNA damage.

17. NUCLEOTIDE EXCISION REPAIR 5 IMPORTANT STEPS 1.Damage recognition 2.DNA incisions that demarcate the lesion(24-32 nucleotides in length) 3.Removal of the region containing the adducts 4.Gap fill up 5.DNA ligation

18. NUCLEOTIDE EXCISION REPAIR

19. DNA-DSB REPAIRS HOMOLOGOUS RECOMBINATION REPAIR 1.Yeast and other lower eukryotes 2.Error free process 3.Requires undamaged DNA template 4.Occurs in late S and G 2 phase 5.Radioresistant in late S phase is due to homologous recombination repair. NONHOMOLOGOUS END JOINING 1.Higher eukaryotes like mammals 2.Error prone 3.Occurs in G 1 phase 4.So G 1 cells are more radiosensitive.

20. NONHOMOLOGOUS END JOINNING IT IS DIVIDED INTO 5 STEPS End recognition by Ku binding Recruitment of DNA dependent protein kinase catalytic subunit End processing Fill in synthesis or end bridging Ligation

21. HOMOLOGOUS RECOMBINATION REPAIR

22. Defect in HRR results in radiosensitivity and genomic instability leading to cancer by loss of heterozygosity

23. CROSSLINK REPAIR Radiation damage produces DNA-DNA and DNA-protein crosslinks Repair mechanism of these crosslinks is still under investigation Probably combination of NER and HRR are needed for the repair Individuals with fanconi’s anemia are hypersensitive to crosslinking agents

24. MISMATCH REPAIR This procedure removes base-base and small insertion mismatches that occur during replication This constitutes four steps Mismatch identification by sensors MMR factors are recruited Incorrect/altered nucleotide are excised Resynthesis and ligation of the excised DNA tract

25. Mismatch identification MMR factor recruitment Incorrect nucleotide excision ligation

26. CLINICAL IMPLICATIONS REPAIR MECHANISM DEFECT DISEASE Nucleotide excision repair Xeroderma pigmentosum Cockayne syndrome Trichothiodystrophy Homologous recombination repair Blooms syndrome Hereditary breast cancer(BRCA 1,BRCA 2 deficiency) Ataxia telangiectasia Nonhomologous end joining Immune deficiency Crosslink repair Fanconi anemia Mismatch repair HNPCC

27. CHROMOSOMAL ABERRATIONS Aberrations seen at metaphase are of 2 types Chromosome aberrations Occurs early in interphase Before replication Chromatid aberrations Occurs in a single chromatid arm after chromosome replication and leaves the opposite arm of the same chromosome undamaged leads to chromatid aberrations.

28. EXAMPLES OF RADIATION INDUCED ABERRATIONS LETHAL ABERRATIONS Dicentrics Rings Anaphase bridge NONLETHAL ABERRATIONS Symmetric translocation Small deletion

29. DICENTRIC CHROMOSOME

30. RING CHROMOSOME

31. ANAPHASE BRIDGE

32. NONLETHAL ABERRATIONS Symmetric Translocation e.g Burkitt lymphoma,leukemia . B. Deletion e.g deletion of tumour suppressor genes leads to carcinogenesis.

33. Chromosomal aberrations in peripheral lymphocytes have been used as biomarkers of radiation exposure Frequency of asymmetric aberrations ( dicentrics and rings) in the lymhocytes reflects the dose received Dose can be estimated by comparision with in vitro cultures exposed to known doses Lethal aberrations are refered to as “unstable” aberrations as their number declines with time Symmetric translocations are refered to as “stable” aberrations because they persist for many years

34. POINTS TO NOTE DSBs are the most relevant lesions leading to most biologic insults from radiation leading to cell killing. Energy deposition by ionizing radiations are in form of spur,blob or short tracks. NHEJ occurs in G 1 phase(radiosensitive) HRR occurs in late S or G 2 phase( radioresistant ) Chromosomal aberrations in peripheral lymphocytes have been used as biomarkers of radiation exposure Effects of radiation on chromosome is described at the first metaphase after radiation