1. Repair of DNA and XP Errol C. Friedberg (2003) DNA damage and repair. Nature 421: 436-440.

2. Xeroderma Pigmentosum (XP) Symptoms include: --- Extreme sensitivity to sunlight --- Early onset of skin cancer

3. NER Healthy DNA Why are XP patients sensitive to sunlight? Healthy DNA UV Damaged DNA NO REPAIR 8 gens

4. Xeroderma Pigmentosum (XP)

5. Hereditary Cancer Syndromes involving defects in DNA Repair Xeroderma pigmentosum..........Nucleotide Excision Repair Hereditary Nonpolyposis......... Colon Cancer Mismatch DNA Repair Ataxia Telangiectasia............... Double-Strand Break Fanconi Anemia DNA..............Crosslink Repair Li-Fraumeni..............................Nucleotide Excision Repair Breast-Ovarian Cancer.............. Double Strand Breaks Nucleotide Excision Repair

6. DNA Damage Metabolism Exogenous Endogenous DNA Replication Permanent Genetic Alteration Disease Cell Cycle Arrest DNA Repair Apoptosis Daño y reparación del DNA

7. Base Loss Base modification & Deamination Chemical Modification Photodamage Inter-strand crosslinks DNA-protein crosslinks Strand breakage Types of DNA damage

8. Abasic site -loss of a nucleobase (apurinic or apyrimidinic) Deamination Base loss

9. Potential Sites of modification/damage

10. Chemical Damage Oxidative damage Alkylation

11. UV-induced damage

13. Types of Damage Repair Photolyase De-alkylation proteins (not catalytic) Base Excision Repair Nucleotide Excision Repair (GG and TC) Recombination Repair Error-prone Repair Double strand Break Repair ( if time permits )

14. UV-responsive photolyases

15. Direct reversal (de-alkylating proteins)

16. Base Excision Repair

18. BER

19. NER

20. Nucleotide Excision Repair (E.coli)

21. Nucleotide Excision Repair (Global Genome Repair -Humans)

22. Nucleotide Excision Repair (Transcription Coupled -Humans)

23. NER

24. Common features of GGR & TCR

25. Mismatch Repair

26. Recombination Repair Other possible mechanisms of recombination repair

27. Mecanismos de Reparación del DNA (DSB) Recombinacion Homologa RAD50 MRE11 NSB1

29. Error Prone Bypass (E. coli)

30. Experimental evidence for Error prone repair (E.coli) Revertant in His- genes (umuC mutated strain) UV-responsive activation of the umuC gene

31. DNA repair polymerases DNA polymerase Eta (XP-V) - addition of two dA residues across pyrimidine dimers DNA polymerase Zeta - addition of random residues across pyrimidine dimers

32. Non-Homologous End Joining (Double Strand Breaks)

33. Model for activation of DNA damage repair

34. Damage & Repair Multiple forms of DNA damage occur These are repaired constantly by several mechanisms Failure to repair damage leads to mutations Often defects in damage sensing machinery or DNA repair processes can be correlated with increased incidence of diseases such as cancer

35. Factors involved in Damage Sensing

38. Apoptosis and DNA Repair Objectives: –1) Understand that programmed cell death or apoptosis is genetically controlled and is an important factor in tumour growth –2) Describe the relationship of abnormal DNA repair to genetic instability and cancer-prone syndromes –3) Describe DNA-damage activated cell-cycle checkpoints and how they prevent mutation and abnormal cellular division

40. Van Gent et al, 2001 TheImportanceofDNA-dsbRepair

41. Molecular DNA-dsb Repair BRCA1/2

42. DNA Damage-Induced Protein Interactions Nbs1Mre11DAPIMerged Time after IR (hrs) 0 0.53612 H2AX BP53 BRCA1 Rad50, Rad51 Indicators or Biomarkers of DNA Repair ?

43. BRCA1/BRCA2 Tumour suppressor genes associated with high risk of breast cancer at young age with bilateral tumour risk (BRCA1 also ovarian and prostate/colon; BRCA2 also ovarian, male breast) These mutations account for 80% of familial breast cancer; yet only 5-10% of breast cancers are familial, the others are sporadic NOT associated with sporadic breast cancer (unlike p53) Both proteins involved in the repair of DNA double strand breaks and predispose to aberrant DNA replication and lead to mutations and cancer

44. ATM BRCA1/2 proteins may have a role in homologous DNA- dsb repair due to ATM, rad50 and rad51 interactions and mutant BRCA1/2 proteins can not interact appropriately

45. HNPPC and Mismatch Repair Hereditary non-polyposis colorectal cancer (HNPPC) is a disorder of faulty mismatch repair and genetic instability evolution of multiple tumours occurs much more rapidly at young age (<50 yrs) and accounts for 2-4% of all colonic cancers Have decreased ability to repair replication errors (RER+ phenotype) due to a lack of the MLH1 or MSH2 genes which normally remove incorrect DNA base pairs Leads to DNA microsatellite instability (MSI) detectable on gel electrophoresis; interestingly MSI+ colon cancers have a better prognosis following therapy

46. Microsatellite Instability (tandom repeats of DNA) is a sign of MMR-deficiency and may be due to MSH2-MLH1 mutations (IHC) = Diagnostic Testing

47. Chromosomal Control and Cancer: Telomeres Cells are capable of only a limited series of divisions before they arrest or senesce and chromosomal fusions and cell death Telomeres caps of chromosome ends and function to prevent DNA loss during DNA replication and provide a cellular clock for cell proliferation They consist of a specific sequence TTAGGGG associated with proteins (TEP, hTERT) As somatic cells normally age, telomeres reduce in length due to decreased function of telomerase, an enzyme which is used by germline cells to maintain telomere length = Cancer Prevention or Tumour Suppression In human tumours, telomerase activity is abnormally high leading to abnormal control of cell growth and proliferation

48. Normal and Abnormal Telomere Functioning In Normal or Cancer Cells

49. Harrington, 2001

50. SUMMARY -Apoptosis is triggered by a number of external stressors including chemo- or radiotherapy and is controlled by the p53, myc and bax-bcl-2 proteins -Distinct morphologic features define apoptotic cells secondary to caspase activity -The relative level of apoptosis versus cell proliferation determines selection of those mutant cells which may proliferate to form a tumour -Defects in DNA repair of UV-damage, DNA replication errors or DNA-dsbs can lead to genetic instability and genetic mutation -These defects lead to cancer-prone syndromes such as xeroderma pigmentosum, HNPPC and BRCA1-associated tumours in which patients are sensitive to specific DNA-damaging agents, develop cancers at an early age and have chromosomal instability in tissues