GENOMIC INSTABILITY AND CANCER: INSIGHTS FROM ANALYSIS OF THE BLOOM’S SYNDROME HELICASE
Ian D. Hickson
CR-UK Oxford Cancer Centre, Weatherall Institute of Molecular Medicine, University of Oxford. PRESENTATION OUTLINE
• How do cancers arise - what genetic changes are necessary?
• Genomic instability and its role in tumorigenesis
• Bloom’s syndrome as a model for understanding how aberrant mitotic recombination can drive tumorigenesis
• A conserved pathway involving the BLM protein and its partners topoisomerase IIIα and BLAP75/RMI1 for resolution of homologous recombination intermediates HOW DO CANCERS ARISE?
1st mutation 2nd mutation 3rd mutation nth mutation
CANCER
• Cancer is a genetic disease - but it is polygenic
• Results from defects in genes that control cell birth and/or cell death
• Cancers acquire several capabilities in order to be life-threatening - evade apoptosis - insensitive to anti-proliferative signals - ability to invade tissue and metastasize - immortal (no replicative limit) THE CLASSIFICATION OF CANCER GENES
• ONCOGENES •Mutation renders gene constitutively active (e.g. Ras) •Drive tumorigenesis e.g. by making cells independent of mitogenic growth signals •One activated allele sufficient
• TUMOUR SUPPRESSOR GENES •Mutation renders gene inactive (e.g. Rb) •Loss of function permits unregulated cell cycle progression etc. •Usually both alleles inactivated Oncogene and TS gene changes directly drive neoplastic transformation by permitting cell proliferation and/or abrogating cell death (GATEKEEPERS)
• GENOME STABILITY GENES (CARETAKERS) •Mutation renders gene inactive •Not necessarily directly involved in or rate limiting for neoplastic transformation •Not selective; simply increase probability that oncogenes/TS genes will be hit •Usually both alleles inactivated •Particularly potent when defect inherited 1st mutation 2nd mutation 3rd mutation nth mutation
CANCER
normal cells malignant cells
Maintenance of Fanconi’s anaemia Bloom’sBloom’s syndromesyndrome (FANCA-M) Genomic Integrity (BLM)(BLM)
Xeroderma Ataxia telangiectasia pigmentosum (ATM) (XPA-G)
Ataxia telangiectasia Nijmegen breakage Hereditary breast -like disorder syndrome and ovarian cancer (MRE11) (NBS1) (BRCA1/2) Bloom’s syndrome
- Autosomal recessive disorder - Short stature - Skin abnormalities - Male infertility/female subfertility - Predisposition to cancer The first 100 Cancers in the Bloom's Syndrome Registry Cases 3 Oesophagus 5 Pharynx 1 Lung 7 Breast 13 Large intestine 1 Metastatic to liver CARCINOMA 11 Skin 3 Larynx 2 Stomach 5 Uterus 2 Hodgkin's disease LYMPHOMA 20 Non-Hodgkin's 14 ANLL LEUKAEMIA 7 ALL 3 Osteogenic sarcoma 2 Wilms's tumor RARE TUMOURS 1 Medulloblastoma 01020304050 Age at Diagnosis CANONICAL DOMAIN ORGANIZATION OF A RecQ HELICASE
Regulatory domain Catalytic ‘core’ NLS
HELICASE RQC HRDC
7 motifs of SF2 helicases DOMAIN STRUCTURE OF SELECTED MEMBERS OF THE RecQ HELICASE FAMILY
E. coli RecQ HELICASE RQC HRDC
TOPO III S.c Sgs1 HELICASE RQC HRDC TOPO III S.p Rqh1 HELICASE RQC HRDC
TOPO III H.s BLM HELICASE RQC HRDC H.s WRN Exo HELICASE RQC HRDC H.s RecQ4 HELICASE H.s RecQ1 HELICASE RQC HRDC H.s RecQ5β HELICASE RQC NLS RecQ HELICASE DEFICIENCY DISORDERS
Werner’s syndrome Rothmund-Thomson syndrome (WRN) (RECQ4) Normal until puberty Congenital skeletal abnormalities Premature ageing Sparse hair and poikiloderma Cataracts Cataracts Cancer prone Cancer prone CANONICAL DOMAIN ORGANIZATION OF A RecQ HELICASE
Regulatory domain Catalytic ‘core’ NLS
HELICASE RQC HRDC
7 motifs of SF2 helicases OVERALL STRUCTURE OF THE CATALYTIC CORE OF E.coli RecQ
Courtesy of Dr James Keck MUTATIONS IN BLM FS: 1226+51-X W428X FS:448+1-X FS: 829+4-X Q752X FS: 514+1-X FS: 802+3-X Y1170X R364X FS: 1195+2-X FS: 750+24-X V1198M FS: 1242+13-X FS: 835+17-X Q1040X FS: 974+23-X R899X D1064V C1066Y FS: 941+26-X H963Y C1055S FS: 1074+4-X FS: 1084+7-X R1139X FS: 1158+5-X Q1283X H1324Y FS: 1009+23-X FS: 1086+10-X S1093X S595X C901Y C1055G C1055R K272X FS: 193+11-X S104X Q672R G891E Q645X G952V FS: 330+3-X L543X Q548X Q909X FS: 656+4-X FS: 91+36-X W567X W881X FS: 256+7-X Q700X FS: 735+4-X S186X
HELICASE RQC HRDC Regulatory domain NLS IVS11: 5'SS Del E11-12 (2) IVS9: 5'SS Del E15 Del E20-22 IVS2: 5’SS IVS6: 3’SS IVS7: 5'SS IVS8: 5'SS IVS16: 5'SS IVS18: 5'SS BLM contains four distinct biochemical activities
• DNA dependent ATPase :
• A 3’-5’ DNA strand displacement activity
BLM 3’ BLM 3’ 3’ 5’ 3’ 5’
• A Holliday junction branch migration activity BLM
• A single stranded DNA annealing activity (ATP independent):
BLM Elevated SCEs are diagnostic of Bloom’s syndrome cells HOW ARE SCEs GENERATED ? ssDNA gap Rad51-mediated strand invasion ‘Early’ alternate pathway (SDSA)
Copying of genetic information from homologous sequence
Endonucleolytic cleavage Gene Conversion DOUBLE HOLLIDAY JUNCTION and rejoining of junctions
Gene Conversion Endonucleolytic cleavage with crossing over ‘Late’ alternate pathway and rejoining of junctions in opposite orientations [SCE] TOPOISOMERASES
• Ubiquitous, highly conserved enzymes • Act to disentangle topological problems that arise during DNA metabolism
Class Gene (S. cerevisiae)
Type 1A TOP3 Type 1 Type 1B TOP1
Type 2 TOP2 How might BLM and topo IIIα catalyze a non- endonucleolytic resolution of double Holliday junctions?
Branch migration BLM?
Double Holliday Junction (Hemicatenane)
Topo III? Generation of a DNA substrate containing two Holliday junctions
R1 H1
* 80-mer oligonucleotides
Anneal oligos
*
1.5 helical turns Ligate nicks
RsaI *
HhaI
11bp 14bp 11bp BLM and hTOPO IIIα cooperate to resolve DHJ
+ + + + + + hTOPO IIIα
B1 RsaI BLM RsaI
HhaI
Requires the ATPase/helicase activity of BLM and the active site tyrosine of topo IIIα BLM and hTOPO IIIα catalyze a novel mechanism to resolve double Holliday Junctions into non-crossover products
hTOPO IIIα-mediated strand passage event
BLM BLM hTOPO IIIα-mediated strand passage Non-crossover event products
Double junction dissolution Double junction dissolution is catalyzed specifically by BLM
BLM WRN BLM RECQ1 BLM RECQ5β
hTOPO IIIα
In contrast, any type IA topoisomerase is functional The C-terminal domain of BLM is required for double junction dissolution
TOPO III Catalytic ‘core’ NLS HELICASE RQC HRDC BLM
HELICASE RQC HRDC BLM-N
HELICASE RQC HRDC BLM-NC
BLM-N BLM BLM-NC
hTOPO IIIα C-terminal truncation of BLM disables the HRDC domain
TOPO III Catalytic ‘core’ NLS
HELICASE RQC HRDC BLM
HELICASE RQC HRDC BLM-N
HELICASE RQC HRDC BLM-NC
Helicase function is not impaired in BLM-NC The HRDC domain of the RecQ helicase family forms an evolutionarily conserved protein-fold
Liu et al, 1999 A third subunit in the BLM/topo IIIα complex
BLAP75, an essential component of BLM-Topo IIIα complex
P I
2 o 5 1 g
o i 7 P o l
I g g i o P l li 5 d o 7
A o M M e
t l 5 P L L a o 7 e r P A
B t B siRNA Oligo r L t A n B n o L U C B
BLM BLAP250 BLAP250 TOPO IIIα
BLM BLM BLAP75
TOPO III TOPO III Beta-actin BLAP75 BLAP75 RPA70 RPA70
BLAP75 is required for the stability of the BLM/topo IIIα complex
Yin et al. EMBO J. 2005 Courtesy of Dr Weidong Wang BLAP75 is conserved in yeast (Rmi1/Nce4)
Sc Rmi1 I II III 241aa
Sp Rmi1 I II III 235aa
Hs BLAP75 I II III 625aa
OB-fold domain hRMI1 strongly stimulates BLM/hTOPOIIIα-dependent dissolution
Time-course - -RMI1 +RMI1
100
80 - hRMI1 60 +hRMI1
40
20 Product (%) 0 0102030405060
Time (min) hRMI1 STIMULATES DISSOLUTION VIA TOPO IIIα
166nM 166nM hRMI1 125nM 125nM hTOPO IIIα 5nM 5nM BLM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14
100 100 - hRMI1 80 +hRMI1 - hRMI1 80 +hRMI1 60 60 40 40 Product (%) Product 20 Product (%) 20
0 0 0.5 1 1.5 2 024681020 40 0 [TOPO IIIα] nM [BLM] nM RMI1 recruits hTOPOIIIα to the double Holliday junction
0 1.5 3 6 12 23 45 90 hTOPO IIIα (nM)
A B
hRMI1
18nM hTOPO IIIα 54nM hTOPO IIIα A B
1 2 3 4 5 6 7 8 9101112131415 1617181920 21 Summary and Additional Data
BLM and hTOPO IIIα catalyze a novel mechanism to resolve recombination intermediates that does not involve RuvC-like endonuclease cleavage (Double junction dissolution)
Double junction dissolution is highly specific for BLM, but requires the action of any type IA topoisomerase
The HRDC domain of BLM (and RecQ) constitutes a DNA structure-specific recognition motif
The HRDC domain of BLM (and Lys-1270) is required for efficient catalysis of dissolution
Double junction dissolution is markedly enhanced by hRMI1 via a stimulation of topo IIIα. hRMI1 binds directly to topo IIIα, and appears to promote its loading to the substrate Alternative template usage during homologous recombination
Resolution into BLM/RMI1/hTOPO IIIα crossover products
sister chromatid chromosome loss of heterozygosity exchange instability
CANCER