1. Immunological diversity Gilbert Chu January 2004

2. Discovery of antibody diversity 430 BCThucydidesOn bubonic plague: ”It was with those who had recovered from disease that the sick and the dying found most compassion. These knew what it was from experience, and had now no fear themselves; for the same man was never attacked twice - never at least fatally.” 1796JennerNoted that cowpox was rarely followed by smallpox Showed that cowpox innoculum protected from smallpox Pasteur coined “vaccine” from vacca, cow in Latin 1901LandsteinerDiscovered antibodies against ABO blood antigens Made antibodies against many organic molecules: specificity and diversity Discovered antibodies against the red blood cell antigen in paroxysmal cold hemoglobinuria: autoimmunity

3. The antibody molecule

4. Mouse immunoglobulin genes V1-V500D1-D12J1-J4 H chain locus (Chr 12) CC CC C3C3 C1C1 C  2bC  2aCC CC V1-V250J1-J5 CC V 2J 2C 2  chain locus (Chr 6) chain locus (Chr 16) C 3C 1V 1J 3J 1

5. Mechanisms for generating antibody diversity l V(D)J recombination l Somatic hypermutation l Class switch recombination V1-V500D1-D12J1-J4constant regions

6. l V(D)J recombination Mechanisms for generating antibody diversity V1-V500D1-D12J1-J4constant regions Hozumi and Tonegawa, PNAS 1976

7. l V(D)J recombination Mechanisms for generating antibody diversity V1-V500D1-D12J1-J4constant regions

8. l V(D)J recombination Mechanisms for generating antibody diversity V1-V500D1-D12J1-J4constant regions

9. l V(D)J recombination D to J joining Mechanisms for generating antibody diversity

10. l V(D)J recombination D to J joining Mechanisms for generating antibody diversity

11. l V(D)J recombination V to DJ joining Mechanisms for generating antibody diversity

12. l V(D)J recombination l Somatic hypermutation ** * Somatic mutations Mechanisms for generating antibody diversity

13. ** * l V(D)J recombination l Somatic hypermutation l Class switch recombination Class switch Mechanisms for generating antibody diversity

14. l V(D)J recombination l Somatic hypermutation l Class switch recombination ** * Class switch Mechanisms for generating antibody diversity

15. l V(D)J recombination l Somatic hypermutation l Class switch recombination ** * V(D)J recombination and class switch recombination involve double-strand breaks Mechanisms for generating antibody diversity

16. Recombination signal sequence (RSS) direct V(D)J recombination

17. V(D)J recombination involves DNA cleavage and end-joining

18. Cleavage is initiated by RAG1/RAG2 (recombination activating genes) van Gent, Gellert et al. Cell 1995

19. DNA ends are modified by addition and deletion l N-nucleotide addition by terminal deoxynucleotidyl transferase (TdT) l P-nucleotide addition by asymmetric opening of hairpin coding ends l Nucleotide deletion

20. Addition at DNA ends

21. Deletion at DNA ends

22. DNA pathways in V(D)J recombination

23. Evolution of V(D)J recombination l RAG1 and RAG2 contain no introns and are tightly linked on the same chromosome l RAG1 and RAG2 are conserved back to the evolution of jawed fish l Evolutionary hypothesis: a transposon with RAG1, RAG2, and associated RSSs infected a precursor of jawed fish

24. RAG1 and RAG2 do not exist in jawless fish hagfish lamprey

25. Hypothetical RAG transposon

26. Transposon integration Agrawal, Eastman and Schatz Nature 1998

27. Origin of the immunoglobulin genes

28. The scid mouse l Mouse with severe combined immunodeficiency, lacking mature B and T cells l Defective in the joining of coding ends Normal in the joining of signal ends l Hypersensitive to ionizing radiation The scid mouse suggested a link between V(D)J recombination and the repair of DNA double-strand breaks

29. Mutant nonlymphoid cells can be tested for V(D)J recombination l Mutagenesis of Chinese hamster epithelial cells generated several X-ray sensitive cell lines l These cells were co-transfected with RAG1, RAG2, and V(D)J recombination substrates l The cells were then assayed for either coding joint formation or signal joint formation

30. V(D)J recombination substrates Lieber, Gellert et al. Cell 1998

31. Mutants in V(D)J recombination and X-ray resistance Taccioli, Alt et al. Science 1993

33. Mechanisms for repairing DNA double-strand breaks l Homologous recombination l Non-homologous end-joining V(D)J recombination mutants are defective in non-homologous end-joining

34. Proteins involved in non-homologous end-joining

35. Human diseases with defects in non-homologous end-joining l Severe combined immunodeficiency with radiation sensitivity (Artemis) l Ataxia telangiectasia-like disorder (Mre11) l Nijmegan breakage syndrome (NBS1)

36. Ku recruits DNA-PKcs to DNA ends DNA-PKcs Ku DNA-PKcs DNA-PKcs then brings DNA ends together

37. Stoichiometry of the synaptic complex

38. Kinase inhibition does not affect synapsis

39. DNA-PK is activated cooperatively by DNA (Phosphorylation occurs after synapsis)

40. Leuther, Hammarsten, Kornberg, and Chu, EMBO J 1999

44. DNA with single-stranded ends activates DNA-PKcs most efficiently Hammarsten, DeFazio and Chu, J Biol Chem 2000

45. DNA ends with single-strand loops fail to activate DNA-PKcs

46. DNA ends blocked with streptavidin fail to activate DNA-PKcs

47. Model for activation of DNA-PKcs

48. Smider, Rathmell, Lieber, and Chu, Science 1994 Non-homologous end-joining

49. Hammarsten and Chu, PNAS 1998 Non-homologous end-joining

50. DeFazio, Stansel, Griffith, and Chu, EMBO J 2002

51. Non-homologous end-joining

53. A jawed fish (trout)

54. Alex’s model for end-joining, 1995

55. Questions about end-joining Protein questions What are the DNA polymerases? What are the nucleases? Phosphorylation questions Which proteins are targeted by DNA-PK? How does phosphorylation regulate these proteins? How does DNA-PK phosphorylate these proteins before phosphorylating itself?

56. Somatic hypermutation (SHM) SHM targets immunoglobulin genes (but not T cell receptor genes) SHM requires active transcription SHM involves DNA single-strand breaks

57. Model for somatic hypermutation Activation-induced deaminase (AID) Expressed only in activated B cells Converts C to U in single-stranded DNA Other proteins insert mutations Uracil DNA glycosylase converts U to an apurinic site AP endonuclease nicks the DNA adjacent to the AP site Exonuclease removes the AP ribose An error-prone polymerase fills in the gap

58. Model for somatic hypermutation How is C mutated on both strands with the same frequency? How does SHM target the Ig locus, but not other loci?

59. Class switch recombination (CSR) CSR rearranges the constant regions to generate different antibody isotypes CSR regions located 5’ to each C H gene, except for C  consist of repeats of GAGCT and GGGGGT; e.g.,  switch region is [(GAGCT) n GGGGGT] 150 CSR requires active transcription AID initiates CSR

60. CSR occurs via double-strand breaks CSR requires Ku and DNA-PKcs CSR junctions show characteristics of non-homologous end-joining Deletions to regions of microhomology Duplications from DNA polymerase activity

61. Model for class switch recombination How does AID initiate CSR at one locus and SHM at another? (The C-terminus of AID is required for CSR, but not SHM.)

62. Summary l Diversity is generated by multiple mechanisms l V(D)J recombination l Somatic hypermutation l Class switch recombination l Some components are lymphocyte-specific l RAG1/RAG2, TdT, AID l Other components are ubiquitous l Double-strand break repair, base excision repair