genomic instability and cancer: insights from
TRANSCRIPT
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
• 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)
HOW DO CANCERS ARISE?
1st mutation 2nd mutation 3rd mutation nth mutation
CANCER
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
• 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
Oncogene and TS gene changes directly drive neoplastic transformation by permitting cell proliferation and/or abrogating cell death (GATEKEEPERS)
Maintenance of Genomic Integrity
Ataxia telangiectasia(ATM)
Fanconi’s anaemia(FANCA-M)
Ataxia telangiectasia-like disorder
(MRE11)
Nijmegen breakage syndrome
(NBS1)
Hereditary breast and ovarian cancer
(BRCA1/2)
Xeroderma pigmentosum
(XPA-G)
Bloom’s syndrome(BLM)
1st mutation 2nd mutation 3rd mutation nth mutation
CANCER
normal cells malignant cells
Bloom’s syndrome(BLM)
Bloom’s syndrome
- Autosomal recessive disorder
- Short stature
- Skin abnormalities
- Male infertility/female subfertility
- Predisposition to cancer
RARE TUMOURS
Age at Diagnosis
Cases
Medulloblastoma1
Metastatic to liver1
Lung1
LYMPHOMA
LEUKAEMIA
CARCINOMASkin11
0 10 20 30 40 50
2 Wilms's tumor
5 Uterus
13 Large intestine
7 ALL3 Osteogenic sarcoma
5 Pharynx3 Oesophagus
7 Breast
3 Larynx2 Stomach
2 Hodgkin's disease20 Non-Hodgkin's14 ANLL
The first 100 Cancers in the Bloom's Syndrome Registry
CANONICAL DOMAIN ORGANIZATION OF A RecQ HELICASE
Regulatory domain NLS
HELICASE RQC HRDC
Catalytic ‘core’
7 motifs of SF2 helicases
DOMAIN STRUCTURE OF SELECTED MEMBERS OF THE RecQ HELICASE FAMILY
HELICASE RQC HRDC
HELICASE RQC HRDC
HELICASE RQC HRDC
HELICASE RQC HRDC
HELICASE RQC HRDC
HELICASE
HELICASE
RQC HRDC
HELICASE RQC
Exo
NLS
E. coli RecQ
S.c Sgs1
S.p Rqh1
H.s RecQ5β
H.s RecQ4
H.s RecQ1
H.s WRN
H.s BLM
TOPO III
TOPO III
TOPO III
Werner’s syndrome (WRN)
Rothmund-Thomson syndrome(RECQ4)
RecQ HELICASE DEFICIENCY DISORDERS
Normal until pubertyPremature ageing
CataractsCancer prone
Congenital skeletal abnormalitiesSparse hair and poikiloderma
CataractsCancer prone
CANONICAL DOMAIN ORGANIZATION OF A RecQ HELICASE
Regulatory domain NLS
HELICASE RQC HRDC
Catalytic ‘core’
7 motifs of SF2 helicases
OVERALL STRUCTURE OF THE CATALYTIC CORE OF E.coli RecQ
Courtesy of Dr James Keck
MUTATIONS IN BLM
C90
1Y
S186
X
K27
2X R36
4XIV
S6: 3
’SS
S595
X
Q67
2RQ
645X
Q70
0XFS
: 735
+4-X
FS: 5
14+1
-X
C10
55S
Del
E11
-12
(2)
IVS2
: 5’S
S
R89
9X
FS: 6
56+4
-X
FS: 9
74+2
3-X
FS: 1
086+
10-X
Q75
2X
G89
1E
S104
XFS
: 91+
36-X
FS: 3
30+3
-X
Q54
8XW42
8X
IVS8
: 5'S
SFS
: 750
+24-
X
W88
1X
FS: 1
074+
4-X
S109
3XR
1139
XFS
: 115
8+5-
XY1
170X
FS: 1
226+
51-X
FS: 1
195+
2-X
FS: 1
009+
23-X
C10
66Y
D10
64V
FS: 1
242+
13-X
IVS7
: 5'S
S
FS: 9
41+2
6-X
Del
E20
-22
FS: 8
02+3
-X
IVS1
8: 5
'SS
FS: 1
93+1
1-X
H96
3Y
FS: 2
56+7
-X
FS:4
48+1
-XL5
43X
W56
7X FS: 8
29+4
-XFS
: 835
+17-
X
Q90
9XG
952V
Q10
40X
C10
55G
C10
55R
Q12
83X
V119
8M
FS: 1
084+
7-X
H13
24Y
IVS9
: 5'S
SIV
S11:
5'S
S
IVS1
6: 5
'SS
Regulatory domain NLS
Del
E15
HELICASE RQC HRDC
BLM contains four distinct biochemical activities
• DNA dependent ATPase :
• A 3’-5’ DNA strand displacement activity
3’ 3’5’ 3’ 3’
5’BLM BLM
• A Holliday junction branch migration activityBLM
• A single stranded DNA annealing activity (ATP independent):
BLM
Elevated SCEs are diagnostic of Bloom’s syndrome cells
HOW ARE SCEs GENERATED ?
DOUBLE HOLLIDAY JUNCTION
Rad51-mediatedstrand invasion
Copying of genetic information from
homologous sequence
ssDNA gap
Gene ConversionEndonucleolytic cleavage and rejoining of junctions
Endonucleolytic cleavage and rejoining of junctionsin opposite orientations
Gene Conversionwith crossing over
[SCE]
‘Early’ alternate pathway(SDSA)
‘Late’ alternate pathway
TOPOISOMERASES
Class
Type 2
Type 1Type 1A
Type 1B TOP1
TOP3
TOP2
Gene (S. cerevisiae)
• Ubiquitous, highly conserved enzymes
• Act to disentangle topological problems that arise during DNA metabolism
How might BLM and topo IIIα catalyze a non-endonucleolytic resolution of double Holliday junctions?
Branch migration
Double Holliday Junction(Hemicatenane)
BLM?
Topo III?
*
Anneal oligos
R1 H1
*
*
Ligate nicks
RsaI
HhaI
1.5 helical turns
Generation of a DNA substrate containing two Holliday junctions
80-mer oligonucleotides
11bp 14bp 11bp
+ + + + + +
BLM
hTOPO IIIα
B1
BLM and hTOPO IIIα cooperate to resolve DHJ
Rsa
I
RsaI
HhaI
Requires the ATPase/helicase activity of BLM and the active site tyrosine of topo IIIα
BLM and hTOPO IIIα catalyze a novel mechanismto resolve double Holliday Junctions into non-crossover products
Double junction dissolution
BLM BLM
hTOPO IIIα-mediatedstrand passage
event
hTOPO IIIα-mediatedstrand passage
event
Non-crossoverproducts
BLM WRN BLM RECQ1 BLM RECQ5β
Double junction dissolution is catalyzed specifically by BLM
hTOPO IIIα
In contrast, any type IA topoisomerase is functional
The C-terminal domain of BLM is required fordouble junction dissolution
BLM BLM-NC
hTOPO IIIα
BLM-N
NLSHELICASE RQC HRDC
Catalytic ‘core’TOPO III
HELICASE RQC HRDC
HELICASE RQC HRDC
BLM
BLM-N
BLM-NC
C-terminal truncation of BLM disables the HRDC domain
Helicase function is not impaired in BLM-NC
NLS
HELICASE RQC HRDC
Catalytic ‘core’TOPO III
HELICASE RQC HRDC
HELICASE RQC HRDC
BLM
BLM-N
BLM-NC
The HRDC domain of the RecQ helicase familyforms 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
BLM
TOPO III
BLAP75RPA70
BLM
TOPO III
BLAP75RPA70
BLAP250BLAP250
BLM
IP
BLA
P75
IP
Yin et al. EMBO J. 2005
TOPO IIIα
BLAP75
Untr
eate
dCo
ntro
l olig
oBL
AP75
olig
o1BL
AP75
olig
o2
Beta-actin
siRNA Oligo
BLM
BLAP75 is required for the stabilityof the BLM/topo IIIα complex
Courtesy of Dr Weidong Wang
BLAP75 is conserved in yeast (Rmi1/Nce4)
I II IIISc Rmi1 241aa
I II IIISp Rmi1 235aa
I II IIIHs BLAP75 625aa
OB-fold domain
0
20
40
60
80
100
0 10 20 30 40 50 60
Time (min)
Prod
uct (
%) - hRMI1
+hRMI1
- -RMI1 +RMI1
Time-course
hRMI1 strongly stimulates BLM/hTOPOIIIα-dependent dissolution
hRMI1 STIMULATES DISSOLUTION VIA TOPO IIIα
0
20
40
60
80
100
0 2 4 6 8 10
[TOPO IIIα]
Prod
uct (
%)
[BLM] nM
hRMI1hTOPO IIIα
BLM
- hRMI1+hRMI1
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
- hRMI1+hRMI1
Prod
uct (
%)
20 40 0.5 1 1.5
nM
2
5nM 5nM
125nM 125nM
166nM 166nM
020406080
100
0
AB
AB
hTOPO IIIα (nM)90452312631.50
18nM hTOPO IIIα 54nM hTOPO IIIα
hRMI1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2120
RMI1 recruits hTOPOIIIα to the double Holliday junction
Summary and Additional Data
BLM and hTOPO IIIα catalyze a novel mechanism to resolve recombinationintermediates that does not involve RuvC-like endonuclease cleavage (Double junction dissolution)
Double junction dissolution is markedly enhanced by hRMI1 via a stimulationof topo IIIα. hRMI1 binds directly to topo IIIα, and appears to promote its loading to the substrate
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-specificrecognition motif
The HRDC domain of BLM (and Lys-1270) is required for efficient catalysis of dissolution
Alternative template usage during homologous recombination
sister chromatid exchange loss of heterozygosity chromosome
instability
Resolution into crossover products
BLM/RMI1/hTOPO IIIα
CANCER