DNA double-strand breaks (DBS)

Irradiation Dna replication

Meiosis and V(D)J recombination

Double strand break

unrepaired

Cell death

Mis-repaired

Chromosomal rearrangements

Carcinogenesis

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

Rag1&Rag2 T-B-SCIDOmenn Syndrome

Ku70&Ku80

DNA-PKcsArtemis

Rs-T-B-SCID TdT, Xrcc4 & Dna ligaseIV

Chromosomal instability Immunodeficiency Growth retardation

SCID T- B-

Rag dependent

Artemis dependent sensitivity to IR

Normal sensitivity to IR

+

hairpin formation

joining

Signal joint

nicking

5'3'

coding joint

5'3'

5'3'

"P" "N" nucleotides

V J

Rag1Rag2

3'3'OH5'

5'OH

5'3' 5'

3'

1) They recognize recombination signal sequences

(RSSs) which flank each coding element;

2) They introduce a double strand break at the border

of the RSS and the coding flank;

RAG FUNCTIONS

3) They process hairpin structures and 3’ overhangs

that are postulated recombination intermediates

NULL MUTATIONS in RAGs

Block of V(D)J recombination process

T-B- SCID

Rags Mutations in Omenn

Partial V(D)J recombination

Omenn syndrome (T+B- scid)

• 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!

OMENN SYNDROMECLINICAL AND LABORATORY FEATURES

Patient 301 Spectratype pre-BMT

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

300bp

225bp

200bp

176bp

300bp

225bp

200bp

176bp

Lane1: Spetratype standard

Lane2: sv1

Lane3: sv2

Lane4: sv3

Lane5: sv4

Lane6: sv5.1

Lane7: sv5.3

Lane8: sv6.1

Lane9: sv6.2

Lane10: sv7

Lane11: sv8

Lane12: sv9

Lane13: sv10

Lane14: sv11

Lane15: sv12

Lane16: sv13

Lane17: sv14

Lane18: sv15

Lane19: sv16

Lane20: sv17

Lane21: sv18

Lane22: sv19

Lane23: sv20

Lane24: sv21

Lane25: sv22

Lane26: sv23

Lane27: sv24

Lane28: Spetratype standard

Legend:

TCRBV6TCRBV1

TCRBV1CTGTGCCAGCA GACAGGGG AATTCACCCC

CTGTGCCAGCAGCGTAG

CTGTGCCAGCAGC

CTGTGCCAGCAGC

CCGG

GGACTAG CTACGAGC

CAGGG CCTG CCTACGAGC

CC GGGG AA CTCCTACGAGC

N D N J

1S62S7

2S7

2S7

CTGTGCCAGCACCCGATTGATCGGGGCCCCACAAGT CACAGATACGC

CTGTGCCAGCAGCT GGACAGGGGG CTACGAGC

CTGTGCCAGCAGCTTA ATTGG TAGCG AGGG CGAGC

TCRBV6 N D N J2S3

2S7

2S7

T CELL REPERTOIRE IN PATIENT M.G. WITH OMENN SYNDROME

Catalytic core

GGRPR

Dna binding domain

RAG 1

D D E

Rag1 N-terminal

Heptamer and nonamer

binding domainRag1 C-terminal

coding DNA binding domain

Coding DNANonamer

SpacerHeptamer

389 GGRPR

INT-homology domain

RAG-1 MUTATIONS IN OS AND IN T- B- SCID

1 1043

R396CR396H

D429G

R561H Y912CR561C

R737H

S401P

A444V

W522C

M458I

E722Kdel 368-369 ->K86fsR507W

E774X

Y938XR897X

L872X

W959X

E719K

Basic domains I, IIa, IIb, III interaction with Srp1

Ring finger domain

Zinc finger domain A

-helix domain DNA binding

Core domain cleavage

Zinc finger domain B catalytic activity

homodimerization

N-terminal domain

INT-homology domain

1 1043

del 368-369 ->K86fs

Which role?

Through the analysis of RAG genes in Omenn patients we detected 7 patients bearing

deletions at RAG1 N-terminal:

OS8 homoz. D368A/369AOS5, OS9-12 heteroz. D368A/369A*OS13 homoz. D887A*the other mutated allele varied between each patient

N-terminal RAG1 role

OS5 del 368 D429G

OS 8 del 368

OS 9 del 368 R624C

OS10 del 368 E722K

OS11 del 368 R829X

OS12 del 368 frameshift

N-terminal Rag1 role

Basic domains

CONCLUSIONS

DELETION AT THE N-TERMINUS OF RAG1 PREDICTS FRAMESHIFTS LEADING TO

TRUNCATED PROTEIN ALLOWING PARTIAL ACTIVITY IN VDJ RECOMBINATION PROCESS

RAG2

Acid regionCatalytic core

RAG2 active core contains six internal repeats of 50 aa identified in the Drosophila kelch protein.

1 382

III

III

IV

V

VI

1273

G95R

RAG2 SIX PROPELLER BLADES

YY 1MSLQMVTVGHNIALIQP GFSLMNFD ……………… GQVFFFGQKG ………………WPKR……………… SCPTGVFHF ……DIKQ……… NHLKLKPA

AA2 IFSKDSCYLPPLRY PATCSYKG SIDSDK HQYIIHGGKT ………………PNN………………… ELSDKIYIM SVACKNNKK VTFRCTEK

AA3 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

-STRAND 1

-STRAND 2

-STRAND 3

LOOP 4-1 LOOP 1-2

LOOP 2-3

LOOP 3-4

-STRAND 4

1 3821 382

Catalytic core

MUTATION OF THE HYDROPHOBIC RESIDUES

1MSLQMVTVGHNIALIQP GFSLMNFD ……………… GQVFFFGQKG ………………WPKR……………… SCPTGVFHF ……DIKQ……… NHLKLKPA

A L2 IFSKDSCYLPPLRY PATCSYKG SIDSDK HQYIIHGGKT ………………PNN………………… ELSDKIYIM SVACKNNKK VTFRCTEK

A L3 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

-STRAND 1

-STRAND 2

-STRAND 3

LOOP 4-1 LOOP 1-2

LOOP 2-3

LOOP 3-4

-STRAND 4

MUTATIONS IN RAG2 Gly-Ser-Thr REGIONS

POINT MUTATIONS IN THE SECOND STRAND

YY 1MSLQMVTVGHNIALIQP GFSLMNFD ……………… GQVFFFGQKG ………………WPKR……………… SCPTGVFHF ……DIKQ……… NHLKLKPA

AA A L2 IFSKDSCYLPPLRY PATCSYKG SIDSDK HQYIIHGGKT ………………PNN………………… ELSDKIYIM SVACKNNKK VTFRCTEK R

AA A L3 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 A A AY L L LA 6 PDWTSDI KHSKIWFG …SNMG… NGTIFLGIPG ……………DNKQAM…………… SEAFYFYTL …RCSEEDL… SEDQKIVS N AQ L

-STRAND 1

-STRAND 2

-STRAND 3

LOOP 4-1 LOOP 1-2

LOOP 2-3

LOOP 3-4

-STRAND 4

MUTATIONS IN HYDROPHOBIC AND GLYCINE-RICH

REGIONS WITHIN THE SECOND -STRAND

Severe effect on VDJ process

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 6 PDWTSDI KHSKIWFG …SNMG… NGTIFLGIPG ……………DNKQAM…………… SEAFYFYTL …RCSEEDL… SEDQKIVS

-STRAND 1

-STRAND 2

-STRAND 3

LOOP 4-1 LOOP 1-2

LOOP 2-3

LOOP 3-4

-STRAND 4

1 382

Catalytic domain

V

MUTANTS IN LOOP REGIONS

R229Q

RAG2 Omenn mutations

Omenn mutations

Variable loop regions of Kelch

repeat

Acid region

Mild effect on VDJ process

MUTANT ALLELES IN 44 PATIENTS WITH

RAG-DEPENDENT IMMUNE DEFICIENCY

MFT

100

Atypical SCID /OS

0

10

20

30

40

50

60

70

80

90

T- B- SCID CID with OS

missense

null mutations

%

*

* including nonsense, frameshift mutations, and deletions

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 patientsPartial Rag activity

No Rag activity

R396C

* Atypical Scid

@Omenn syndrome

A444V * Atypical SCID

@Omenn syndrome

R229WR229Q

* Atypical SCID

@Omenn syndrome

* Atypical SCID

@Omenn syndrome

R561H

+

hairpin formation

joining

Signal joint

nicking

5'3'

coding joint

5'3'

5'3'

"P" "N" nucleotides

V J

Rag1Rag2

3'3'OH5'

5'OH

5'3' 5'

3'

Artemis

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)

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

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

Rag1 Rag2

Hairpin coding ends

Signal ends

Coding joint Signal joint

PKA

A

TdT

Rag1Rag2

PK

Rag2Rag1OH

HO

NNNNNNNNNN

Coding joints

Hairpin opening

1 2 3 4 5 6 7 98 10 11 12 13 14

Metallo lactamase caspase

*S119X I16T

Artemis as genomic care taker

0,001

0,01

0,1

1

10

100

0 1 2 3 4 5

Gy

Su

rviv

al

CT

ATGS

PT 5

PT 4

PT 3

PT 2

PT 1

0

25

50

75

100

0 1 3 5

MMC

Su

rviv

al

CT

PT 5

PT 4

PT 2

Pt 3

XP-F

PT 1

1BR3

0

25

50

75

100

0 2 4 6

Cisplatin

Su

rviv

al NIG

Melis

Zengin

Infante

Testa

XP-F

Artemis and genomic instability

Anna VillaAntonio Musio

Veronica MarrellaPaolo Vezzoni

CNR-LITAMilano, Italy

L.Notarangelo

S.Giliani

S.Santagata

E.Spanopolou

P. Cortes