DNA Repair 1 : Types of DNA damage

1. DNA Repair 1 : Types of DNA damage Radiobiology 2012

2. Repair of DNA damage caused by ionizing radiation (IR) is defined by the lesion to be repaired: • excision repair • single strand break (SSB) repair • double-strand break (DSB) repair • other repair types (ie crosslink) • DNA Damage and Chromosomal Damage

3. Sources of DNA damage • UV damage to skin • Replication errors generate mismatches • Spontaneous cytosine deamination • Replication fork collapse and strand breaks • Ionizations from high energy photons/particles

4. Ionizing radiation ionizes along tracks

5. LET is linear energy transfer

6. Different LET radiations have different toxicities

7. LET can modify RBE (relative biologic effect) Maximum RBE at 100KeV/uM

8. DNA damage is a complex set of lesions, but things can be simplified: Outcomes of DNA repair: Accurate repair: cell survives without mutations Misrepair: cell survives but at the cost of genetic changes • Inadequate repair: • cell inactivation or • death due to • mitotic death • apoptosis • permanent arrest

9. Special IR Feature: Clustered Damage 2 nm up to ~ 20 bp Spur 4 nm Repair of such a multiply damaged site may create DSBs

10. MMR Genes and Cancer • Hereditary Non-Polyposis • Colon Cancer (HNPCC) • MSH2 • MLH1 • PMS1, PMS2 • Sporadic Colon Ca • MSH2 • MLH1 • Sporadic Endometrial Ca • MSH2 • MSH3 Marti, J Cell Phys 2002

11. IR-induced DNA Damage is heterogeneous Damage typeNo./Gy/cell base damage > 1000 single-strand 500-1000 break (SSB) double-strand ~ 40 break (DSB) sugar damage, various DNA-DNA and DNA- protein cross links

12. C-T-U-A-T G-A-G-T-A Base Excision Repair (BER) DNA glycosylase AP endonuclease (APE) and phosphodiesterase DNA polymerase b adds in “C” and DNA ligase III seals the nick C G

13. BER and Radiation Sensitivity • IR-induced base damage is efficiently repaired • Defects in BER may lead to an increased mutation rate but usually do not result in cellular radiation sensitivity • However, one exception is mutation of the XRCC1 gene (X-Ray Cross Complementation factor 1), which confers ~ 1.7-fold increased radiation sensitivity

14. Functions of XRCC1 XRCC1 PARP-1 Recognition of damage Ligase III Repair of nick XRCC1 Radiation sensitivity of XRCC1-deficient cells may come from XRCC1’s involvement in other repair pathways, such as the repair of SSBs ....

15. Nucleotide Excision Repair (NER) UV IR pyrimidine dimer SSB Helicase Nuclease Polymerase Ligase

16. NER and Radiation Sensitivity • IR-induced SSBs are efficiently repaired • Mutated NER genes do not cause cellular radiation hypersensitivity • However, persistent adjacent SSBs may lead to DSBs & thereby to cell death • Defective NER increases sensitivity to UV-induced damage and to other lesions that affect a single strand • Germline mutations in NER genes cause human DNA repair deficiency disorders XP CS TTD

17. Bulky lesions such as UV damage NER: Global Genome Repair (GGR) and Transcription-Coupled Repair (TCR) GGR TCR defective in Xeroderma Pigmentosum(XP) = repair of transcribed strand in active genes, defective in Cockayne’s Syndrome (CS) and in XP

18. Functions of XP Genes helix unwinding TFIIH XPD HHR23B damage recognition XPB RPA XPC XPA DNA binding factors XPG XPF ERCC1 strand incision • XPC is only required for GGR - not for TCR • function of CSA and CSB is not well understood

19. XPD K751Q polymorphism

20. The Repair of DSBs Why do we believe that a DSB is the most important type of DNA damage induced by IR?

21. DSE vs DSB Nickloff et al Cell Res 2008

22. Defective DSB Repair causes cellular &clinical Radiation Hypersensitivity 14-year old boy with ALL overreacted to radiation therapy and was found to have a mutation in the Ligase IV gene. Riballo, Curr Biol 1999 and JBC 2001 LigIV+/+ LigIV-/- LigIV+/- Grawunder, Mol Cell 1998

23. Measuring DNA Double-Strand Breaks 1. Nucleoid sedimentation • Irradiate cells (100Gy) • Lyse cells and layer DNA on a sucrose gradient (5 - 20%) • Centrifuge at high speed • Collect fractions and measure DNA/fraction • As amount of breaks > density sedimentation > Irradiated control DNA content Fraction sedimented

24. Measuring DNA Double-Strand Breaks Neutral elution (pH = 7.4) Alkaline elution for SSB (pH = 12.2) • Irradiate cells • Lyse cells on filter • Vacuum elute in neutral pH buffer • Collect eluted buffer and measure amount of DNA • As # of breaks > amount of DNA eluted from filter > 0Gy 5Gy % DNA retained 10Gy 20Gy Fraction number

25. Measuring DNA Double-Strand Breaks Electrophoretic - Comet assay, pulsed field electrophoresis

26. Measuring defective DSBR Pulsed-field gel electrophoresis: FAR = fraction of activity released 180BR = LIG4 mutation MRC5 = control filled circles/squares: transformed clones Badie, Cancer Res 1997

27. DSBs can lead to Chromosome Aberrations Immediate Outcomes: 1) No repair: loss of chromosomal end 2) Re-joining of ends, but with change of sequence 3) Joining of ends with other breaks/chromosomes Cell Fate: 1) Survival with genetic changes 2) Apoptosis 3) Mitotic death due to lethal chromosomal aberrations 4) Delayed post-mitotic death or inactivation

28. Type of cytogenetic damage observed depends upon where in the cell cycle irradiation occurs • CHROMOSOME ABERRATIONS • G1 irradiation • Both sister chromatids involved • CHROMATID ABERRATIONS • S or G2 irradiation • Usually only 1 chromatid involved

29. Multiple mis-rejoining events occurring in CHO chromosomes after G1 irradiation tricentric dicentrics

30. Chromatid deletions in CHO chromosomes after irradiation in S or G2 Iso-chromatid deletion Chromatid deletion

31. Combinatorial “painting” - limited use for rare events Spectral karyotying (Sky) m-FISH after irradiation From: Dr. M. Cornforth

32. Inadequate DSB Repair may contribute to Carcinogenesis Chromosome aberrations Small mutations at break site Genomic instability Mutation of oncogenes and tumor suppressor genes e.g., loss of checkpoint control, apoptotic response Malignant cell transformation

33. Why are there two principal Pathways of DSB Repair ?

34. DSB Repair by Homologous or Non-Homologous Recombination (HR, NHR) NHR HR

35. Gene Conversion Model of HR " "

36. HR is essential for DNA Replication Haber, TBS 1999 The HR pathway probably has arisen to repair - spontaneous breaks that occur during replication - broken replication forks in order to restart replication

37. Execution of HR end processing homology search Rad52 single-strand invasion Rad51 + paralogs, Rad54, RPA, BRCA2

38. Uncontrolled HR may be detrimental Mechanisms of Loss of Heterozygosity (LOH): gene conversion normal cell heterozygous cell deletion Up-regulated or de-regulated HR is likely an important mechanism in carcinogenesis. chromosome loss

39. Effects of defective HR 1. Impaired ability to repair DNA in S and G2 phase 2. Cellular hypersensitivity to IR (variable) 3. Often reduced proliferation (because of impaired DNA replication) 4. Chromosomal instability & cancer predisposition: - BRCA2 +/- (familial breast ca & others) - BRCA1 +/- (familial breast ca, ovarian & others) - BRCA1 hypermethylation (sporadic breast ca) - mutations of Rad52, Rad54, XRCC3 and other HR genes found in various sporadic cancers

40. NHR is error-prone Error-prone repair of DSBs by NHR Intentional diversity during V(D)J recombination Mammalian genomes may tolerate error-prone NHR, because > 90% of the DNA sequence is non-coding.

41. (1) (2) (3) (4) NHR is needed for V(D)J Recombination (4) (5a) (5b) (6) CE, coding ends SJ, signal joints Grawunder & Harfst, Curr Opin Immun 2001

42. DNA-PKcs Artemis XRCC4 Ligase IV Enzymology of NHR Ku70/80

43. Effects of defective NHR 1. Impaired ability to rejoin DNA ends 2. Cellular hypersensitivity to IR 3. Impaired V(D)J recombination ® immune defect For example: SCID (severe combined immune deficiency syndrome) 4. Cancer predisposition in mice; however, NOT (yet) linked to human cancer predisposition (except for one leukemia pt with LIG4 mutation) 5. Developmental defects

44. Principal Effects of defective HR or NHR Endpoint HR NHR Chromosomal + + aberrations (esp. chromatid !) Proliferative defect + - Immune defect - + IR sensitivity + ++ Cellular/clinical phenotype varies with particular gene defect

45. Other Types of IR-induced Damage • Damage to sugar back bone • frequent IR damage • easily repaired by excisional repair mechanisms • DNA-DNA long intra-strand and inter-strand cross-links • DNA-protein cross-links • Repaired by mixture of repair mechanisms • Role for radiation sensitivity unclear • Important lesions caused by certain chemotherapeutic drugs (cisplatinum)

46. Therapeutic Potential ? Mitomycin C 5-13% ICLs Cisplatin 5-8% ICLs Topoisomerase I + II inhibitors DSBs - CPT11 - Etoposide Combination with IR: - additive/synergistic cell killing by increasing DSB burden ? - targeting tumors with certain defects in recombination ?

47. Summary of Key Points • IR creates a heterogeneous spectrum of DNA lesions • DSBs constitute the most dangerous type of damage • IR sensitivity correlates best with DSBs • Multiple pathways of DNA repair exist, including BER, NER, HR, NHR, MMR • Inadequate DSB repair can either lead to - cell death/inactivation (due to chromosome aberrations or apoptosis), or to • - carcinogenesis (due to chromosome aberrations or • an increased rate of small mutations)