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DNA double-strand breaks (DBS)

DNA double-strand breaks (DBS). Irradiation Dna replication Meiosis and V(D)J recombination. Double strand break. unrepaired. Mis-repaired. Carcinogenesis. Cell death. Chromosomal rearrangements. Repair Pathways. Homologous recombination which functions in late S/G2

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DNA double-strand breaks (DBS)

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  1. DNA double-strand breaks (DBS) Irradiation Dna replication Meiosis and V(D)J recombination Double strand break unrepaired Mis-repaired Carcinogenesis Cell death Chromosomal rearrangements

  2. Repair Pathways • Homologous recombination which functions in late S/G2 • DNA non-homolgous end-joining (NHEJ)which plays the major role in the repair of radiation-induced DSBs

  3. T-B-SCID OmennSyndrome Rag1&Rag2 Ku70&Ku80 DNA-PKcs Artemis TdT, Xrcc4 & Dna ligaseIV Rs-T-B-SCID Chromosomal instability Immunodeficiency Growth retardation

  4. Normal sensitivity to IR Rag dependent SCID T- B- sensitivity to IR Artemis dependent

  5. Rag2 Rag1 nicking OH 3' 5' 5' 3' OH hairpin formation 5' 3' 3' 5' joining "P" "N" nucleotides 5' 3' coding joint Signal joint V J 3' 5' 5' 3' +

  6. RAG FUNCTIONS 1) Theyrecognize recombination signal sequences (RSSs) which flank each coding element; 2) They introduce adouble strand breakat the border of the RSS and the coding flank; 3) They process hairpin structures and 3’ overhangs that are postulated recombination intermediates

  7. NULL MUTATIONS in RAGs Block of V(D)J recombination process T-B- SCID

  8. Rags Mutations in Omenn Partial V(D)J recombination Omenn syndrome(T+B- scid)

  9. OMENN SYNDROME CLINICAL AND LABORATORY FEATURES • Early-onset erythrodermia (GvH-like) • Lymphadenopathy • Hepatosplenomegaly • Severe hypoprotidemia • Eosinophilia • Lack of circulating B cells • Hypogammaglobulinemia, but elevated IgE • Presence of activated, anergic autologous T cells* • Lethal, unless treated with BMT • *presence of maternally-engrafted T cells must be ruled out!

  10. Legend: Lane1: Spetratype standard Lane8: svb6.1 Lane15: svb12 Lane22: svb19 Lane2: svb1 Lane9: svb6.2 Lane16: svb13 Lane23: svb20 Lane3: svb2 Lane10: svb7 Lane17: svb14 Lane24: svb21 Lane4: svb3 Lane11: svb8 Lane18: svb15 Lane25: svb22 Lane5: svb4 Lane12: svb9 Lane19: svb16 Lane26: svb23 Lane6: svb5.1 Lane13: svb10 Lane20: svb17 Lane27: svb24 Lane7: svb5.3 Lane14: svb11 Lane21: svb18 Lane28: Spetratype standard Patient 301 Spectratype pre-BMT 300bp 300bp 225bp 225bp 200bp 200bp 176bp 176bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

  11. T CELL REPERTOIRE IN PATIENT M.G. WITH OMENN SYNDROME TCRBV6 TCRBV1 TCRBV1 N D N J TCRBV6 N D N J 2S3 CTGTGCCAGCA CCCGATTGATC GGGGC CCCACAAGT CACAGATACGC 1S6 CTGTGCCAGCA GACAGGGG AATTCACCCC 2S7 2S7 CTGTGCCAGCAGCT GGACAGGGGG CTACGAGC CTGTGCCAGCAGCGTAG GGACTAG CTACGAGC 2S7 2S7 CTGTGCCAGCAGC CCGG CAGGG CCTG CCTACGAGC CTGTGCCAGCAGCTTA ATTGG TAGCG AGGG CGAGC 2S7 CTCCTACGAGC CTGTGCCAGCAGC CC GGGG AA

  12. RAG 1 Dna binding domain Catalytic core D D E GGRPR

  13. Rag1 N-terminal Heptamer and nonamer binding domain 389 GGRPR Rag1 C-terminal coding DNA binding domain Nonamer Coding DNA Spacer Heptamer

  14. Basic domains I, IIa, IIb, III interaction with Srp1 Ring finger domain homodimerization Zinc finger domain A a-helix domain DNA binding Core domain cleavage Zinc finger domain B catalytic activity RAG-1 MUTATIONS IN OS AND IN T- B- SCID R507W S401P L872X E722K W522C del 368-369 ->K86fs D429G E774X R897X E719K R737H A444V R396C R561C Y938X M458I Y912C W959X R396H R561H 1 1043 INT-homology domain

  15. N-terminal domain del 368-369 ->K86fs 1 1043 INT-homology domain Which role?

  16. Through the analysis of RAG genes in Omenn patients we detected 7 patients bearing deletions at RAG1 N-terminal: OS8 homoz. D368A/369A OS5, OS9-12 heteroz. D368A/369A* OS13 homoz. D887A *the other mutated allele varied between each patient

  17. N-terminal RAG1 role OS5 del 368 D429G OS 8 del 368 OS 9del 368 R624C OS10 del 368 E722K OS11 del 368 R829X OS12 del 368 frameshift

  18. N-terminal Rag1 role Basic domains

  19. CONCLUSIONS DELETION AT THE N-TERMINUS OF RAG1 PREDICTS FRAMESHIFTS LEADING TO TRUNCATED PROTEIN ALLOWING PARTIAL ACTIVITY IN VDJ RECOMBINATION PROCESS

  20. RAG2 Catalytic core Acid region 1 382 RAG2 active core contains six internal repeats of 50 aa identified in the Drosophila kelch protein.

  21. V 1273 IV VI III I II G95R RAG2 SIX b-PROPELLER BLADES

  22. Catalytic core 1 382 382 1 LOOP 1-2 b-STRAND 1 b-STRAND 2 LOOP 2-3 LOOP 4-1 b-STRAND 4 LOOP 3-4 b-STRAND 3 YY 1MSLQMVTVGHNIALIQP GFSLMNFD ……………… GQVFFFGQKG ………………WPKR……………… SCPTGVFHF ……DIKQ……… NHLKLKPA AA 2 IFSKDSCYLPPLRY PATCSYKG SIDSDK HQYIIHGGKT ………………PNN………………… ELSDKIYIM SVACKNNKK VTFRCTEK AA 3 DLVGDVPEPRY GHSIDVVY …SRGK… SMGVLFGGRS YMPSTQRTTEKWNSVA DCLPHVFLI …DFEFGCA… TSYILPEL FA 4 QDGLSF HVSIARN ………………… DTVYILGGHS ………………LASN……………… IRPANLYRI RVDLPLGT… PAVNCTVL AFAA AA 5 PG GISVSSAI LTQTNN… DEFVIVGGYQ ………………LENQ……………… KRMVCSLVS ……LGDNT…… IEISEMET AY 6 PDWTSDI KHSKIWFG …SNMG… NGTIFLGIPG ……………DNKQAM…………… SEAFYFYTL …RCSEEDL… SEDQKIVS

  23. MUTATION OF THE HYDROPHOBIC RESIDUES

  24. LOOP 1-2 b-STRAND 1 b-STRAND 2 LOOP 2-3 LOOP 4-1 b-STRAND 4 LOOP 3-4 b-STRAND 3 1MSLQMVTVGHNIALIQP GFSLMNFD ……………… GQVFFFGQKG ………………WPKR……………… SCPTGVFHF ……DIKQ……… NHLKLKPA A L 2 IFSKDSCYLPPLRY PATCSYKG SIDSDK HQYIIHGGKT ………………PNN………………… ELSDKIYIM SVACKNNKK VTFRCTEK A L 3 DLVGDVPEPRY GHSIDVVY …SRGK… SMGVLFGGRS YMPSTQRTTEKWNSVA DCLPHVFLI …DFEFGCA… TSYILPEL A L 4 QDGLSF HVSIARN ………………… DTVYILGGHS ………………LASN……………… IRPANLYRI RVDLPLGT… PAVNCTVL A L 5 PG GISVSSAI LTQTNN… DEFVIVGGYQ ………………LENQ……………… KRMVCSLVS ……LGDNT…… IEISEMET 6 PDWTSDI KHSKIWFG …SNMG… NGTIFLGIPG ……………DNKQAM…………… SEAFYFYTL …RCSEEDL… SEDQKIVS

  25. MUTATIONS IN RAG2 Gly-Ser-Thr REGIONS

  26. POINT MUTATIONS IN THE SECOND b STRAND

  27. LOOP 1-2 b-STRAND 1 b-STRAND 2 LOOP 2-3 LOOP 4-1 b-STRAND 4 LOOP 3-4 b-STRAND 3 YY 1MSLQMVTVGHNIALIQP GFSLMNFD ……………… GQVFFFGQKG ………………WPKR……………… SCPTGVFHF ……DIKQ……… NHLKLKPA AAA L 2 IFSKDSCYLPPLRY PATCSYKG SIDSDK HQYIIHGGKT ………………PNN………………… ELSDKIYIM SVACKNNKK VTFRCTEK R AA A L 3 DLVGDVPEPRY GHSIDVVY …SRGK… SMGVLFGGRS YMPSTQRTTEKWNSVA DCLPHVFLI …DFEFGCA… TSYILPEL FA A L 4 QDGLSF HVSIARN ………………… DTVYILGGHS ………………LASN……………… IRPANLYRI RVDLPLGT… PAVNCTVL AFAAPPAA AA A L 5 PG GISVSSAI LTQTNN… DEFVIVGGYQ ………………LENQ……………… KRMVCSLVS ……LGDNT…… IEISEMET DAA AY L L LA 6 PDWTSDI KHSKIWFG …SNMG… NGTIFLGIPG ……………DNKQAM…………… SEAFYFYTL …RCSEEDL… SEDQKIVS NAQ L

  28. MUTATIONS IN HYDROPHOBIC AND GLYCINE-RICH REGIONS WITHIN THE SECOND b-STRAND Severe effect on VDJ process

  29. Catalytic domain 1 382 LOOP 1-2 b-STRAND 1 b-STRAND 2 LOOP 2-3 LOOP 4-1 b-STRAND 4 LOOP 3-4 b-STRAND 3 V 1MSLQMVTVGHNIALIQP GFSLMNFD ……………… GQVFFFGQKG ………………WPKR……………… SCPTGVFHF ……DIKQ……… NHLKLKPA 2 IFSKDSCYLPPLRY PATCSYKG SIDSDK HQYIIHGGKT………………PNN………………… ELSDKIYIM SVACKNNKK VTFRCTEK R* 3 DLVGDVPEPRY GHSIDVVY …SRGK… SMGVLFGGRS YMPSTQRTTEKWNSVA DCLPHVFLI …DFEFGCA… TSYILPEL 4 QDGLSF HVSIARN ………………… DTVYILGGHS………………LASN……………… IRPANLYRI RVDLPLGT… PAVNCTVL 5 PG GISVSSAI LTQTNN… DEFVIVGGYQ ………………LENQ……………… KRMVCSLVS ……LGDNT…… IEISEMET D 6 PDWTSDI KHSKIWFG …SNMG… NGTIFLGIPG ……………DNKQAM…………… SEAFYFYTL …RCSEEDL… SEDQKIVS

  30. MUTANTS IN LOOP REGIONS

  31. R229Q

  32. RAG2 Omenn mutations Omenn mutations Variable loop regions of Kelch repeat Acid region Mild effect on VDJ process

  33. missense null mutations MUTANT ALLELES IN 44 PATIENTS WITHRAG-DEPENDENT IMMUNE DEFICIENCY 100 90 % 80 70 60 50 40 * 30 20 10 0 T- B- SCID CID with Atypical SCID /OS OS MFT including nonsense, frameshift mutations, and deletions *

  34. MOLECULAR FINDINGS We can consider three groups: 1) T-B-SCID patients (no T and B cells) 2) Atypical T-B- SCID patients ( few T and/or B cells) 3) Omenn patients No Rag activity Partial Rag activity

  35. R396C @Omenn syndrome * Atypical Scid * Atypical SCID @Omenn syndrome R561H * Atypical SCID @Omenn syndrome A444V * Atypical SCID @Omenn syndrome R229W R229Q

  36. Rag2 Rag1 nicking OH 3' 5' 5' 3' OH hairpin formation 5' 3' 3' 5' joining "P" "N" nucleotides 5' 3' coding joint Signal joint V J 3' 5' 5' 3' Artemis +

  37. Artemis • Greek goddess Artemis, a guardian of young children and small animals • J.P de Villartay and Despina Moshous discovered Artemis as the gene responsible for RS-T-B-SCID (Cell 105,2001)

  38. Humans Mice and Genomic instability • Artemis patients show defects in coding joint formation but not signal joints- defects in hairpin opening • KO Mice show defects in coding joints, murine fibroblasts have striking genomic instability

  39. Artemis as genome guardian • D.Moshous described lymphoma after EBV infection in patients with hypomorphic Artemis mutations • Artemis is also important for general DNA damage repair

  40. Rag1 Rag2 Hairpin coding ends Signal ends Signal joint Coding joint

  41. Hairpin opening A PK Rag2 Rag1 A PK OH Rag1 HO Rag2 TdT NNNNN NNNNN Coding joints

  42. S119X * I16T 12 1 2 3 4 5 6 7 8 9 10 11 13 14 Metallo b lactamase b caspase

  43. Artemis as genomic care taker

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