A multiprotein complex in DNA damage response network of Fanconi anemia, Bloom syndrome and Breast cancer Weidong Wang Lab of Genetics, NIA
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A multiprotein complex in DNA damage response network of Fanconi anemia, Bloom syndrome and
Breast cancer
Weidong WangLab of Genetics, NIA
A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia
Bloom Syndrome
. Genomic Instability:-sister-chromatid exchange
. Cancer predisposition
. Mutation in BLM, a RecQ DNA Helicase
. BLM participates in:HR-dependent DSB repairRecovery of stalled replication forks
. BLM works with Topo IIIa and RMI to Suppress crossover recombination
Courtesy of Dr. Ian Hickson
A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia
kDa
200-
116-97-
66-
45-
30-
20-
12-
BLM*FANCA*
TOPO IIIα*
RPA 70*
RPA32*
RPA14*
Con
tro l
I P
BL
M IP
IgG H
IgG L
BLAP 250
MLH1*BLAP 75
BLAP 100
Meetei et al. MCB 2003
HeLaNuclear Extract
BLM IP
A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia
RPA70*/FANCG*
FANCC*/FANCE*
BL
M IP
FAN
CA
IP
TOPO IIIα*
RPA 70*
BLM*
FANCA*
IgG L
IgG H
FANCA*
FAAP 100FAAP 95
FAAP 43FANCF*
BLAP 100
BLAP 75MLH1
RPA32*
BLM*
TOPO IIIα*
BLAP 75
FAAP 250 BLAP 250
Meetei et al. MCB 2003Meetei et al. Nat Genet. 2003, 2004, 2005
FANCM=
FANCB=
FANCL=
HeLaNuclear Extract
BLM IPFANCA IP
BRAFT-a Multisubunit Machine that Maintains Genome Stability and is defective in Fanconi anemia and Bloom syndrome
FANCAFANCCFANCEFANCFFANCG
FANCM
FANCB
FANCL
BRAFT Super-complex
BLM
Topo IIIα
BLAP75RMI1
RPA70
Fanconi AnemiaCore Complex
Bloom SyndromeComplex
FAAP100
RPA34
RPA14
12 polypeptides 7 polypeptidesHelicase (HJ, fork, D-loop), fork regression, dHJ dissolution
Topoisomerase, dHJ dissolution
Ubiquitin ligaseBinds and translocatesBranch point (4 WJ, fork)dsDNA translocase
Stimulates dHJ dissolution.Mediates protein-proteininteractions
Binds ssDNAStimulates BLM helicaseStimulates dHJ dissolutionFAAP24Targets FANCM to
ssDNA MHF1MHF2Targets FANCM to
dsDNA
RMI2
BLAP250
Fanconi Anemia: A multi-Gene Disease Model to Study Repair of Crosslinked DNA Damage and Function of BRCA Proteins
• 13 complementation groups and genes– FANC-A,B,C,D1,D2,E,F,G,I, J, L,M,N
• BRCA1 and BRCA2 connection– BRCA2=FANCD1– PALB2=FANCN– BACH1/BRIP1=FANCJ
• Cellular Hypersensitivity to DNA- crosslinking drugs
– A model for studying repair of crosslinked DNA damage
• Genomic Instability: – Chromosomal breaks and interchanges
• Cancer predisposition: – Myeloblastic leukemia– Squamous cell carcinomas
• Aging:– Bone Marrow Failure– Skin abnormalities—look older
Courtesy of Dr. Johan de Winter
FANCA
FANCBFANCCFANCEFANCF
FANCG
FANCL
Group I:FA CoreComplex
Ring Finger
FANCM XERCC4-likeHelicase
TPR motifs
XFAAP24
FAAP100Coiled-coil
Coiled-coil
P
P
P
P
MHF1 &MHF2
ELF DRWD
Histone-fold
Group I FA proteins constitute the FA core complex that has a E3 ubiquitin ligase and a DNA remodeling enzyme
E3 ub ligase
DNA binding& remodeling
Group II FA proteins are monoubiquitinated by the FA core complex; Group III may act downstream of the FA pathway
FANCD2
FANCD1(BRCA2)
FANCJ(BRIP1)
Helicase
BRC Repeats
Group III:BRCA1association
FANCI
FANCN(PALB2)
WD40 Repeats
PartnersGroup II:FA ID complex
UbPARM repeats
ARM repeats
OB-fold DNA binding
UbP
P
P
Partners
ATR
p
D1(BRCA2)
N (PALB2)
J (BACH1)
p
p
FA pathway:
Switch
Fanconi anemia core complex participates in monoubiquitinationof FA ID complex through FANCL and DNA repair via FANCM
USP1
Xue HMG 2008
MHF
Vertebrate FANCM interacts with multiple repair and signaling partners to integrate signals from several pathways
Helicase ERCC4MM1 MM2
FAAP24MHF FA Core
Ub-FANCD2Ub-FANCI
UbBind ssDNA
HCLK2
ATR
Branch pointRemodeling
dsDNA
ATRCheckpoint(Chk1-P)
BLMTop3/RMI
dHJ dissolutionHR repairFork reversal
Repair of Interstrand CrosslinksSCE suppression
Binds branch point(HJ, Fork)
FANCM
FANCM-MHF may represent a minimal version of the FA core complex conserved in all eukaryotes
FANCM/MPH1/FML1
BLMSGS1
Topo IIIα
BLAP75RMI1MHF1
MHF2
Fanconi AnemiaCore Complex
BLM/Sgs1Complex
Yeast and all eukaryotes
??
MHF1 and MHF2 Are New Components of the FA Core Complex
MHF2
FANCM
FANCA
TOPIIIα
FANCGFANCC&E
FANCF
FAAP24
MHF1
BLAP75
BLM
RPA70
RPA32
FAAP100
FANCM IP
200
11697
6655
36
31
21
146
MHF1/2: FANCM -associated Histone Fold protein 1 and 2.
HeLaNuclear Extract
Superose 6fractionation
FANCM IP
(Yan et al. Mol Cell in press)
MHF1 and MHF2 Are New Components of the FA Core Complex
MHF2
FANCM
FANCA
TOPIIIαFAAP100
FANCGFANCC&E
FANCF
FAAP24
BLM
BLAP75
RPA32
RPA70
MHF1
MHF1 IP
200
11697
6655
36
31
21
146
FA-core complex
BLM complex
A
G
B
C
E
F
L100
FANCM 24
BLM
TopIIIα
RMI1RMI2RPA
MHF
FANCM-associated Proteins
HeLaNuclear Extract
Superose 6fractionation
MHF IP
MHF1 and MHF2 can also form a complex without FANCM
22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52
BRAFT MHF
FANCM
MHF1MHF2
kDa670 440 232 158
MHF1 IP
MHF1MHF2
200
116976655
363121146
MHF2
MHF1
XHIS
-MHF1
HIS-M
HF2HIS
-MHF c
omple
x
MHF1/CENP-S
MHF2/CENP-X α1 α2 α3 81 aa
α1 α2 α3 138 aa
Histone-fold MHF complex
(CENP-S complex)
MHF1 MHF2
MHF is identical to the CENP-S complex and Binds dsDNA
MH
F
MH
F2M
HF1 MH
F
MH
F2M
HF1
__
* *ssDNA dsDNA
*MHF
Amano et al. JCB 2009
MHF provides a complementary DNA binding interface for FANCM-FAAP24 to recognize stalled forks
Helicase ERCC4
MHFFANCM
FAAP24
ssDNAdsDNABranch
point
FANCMMHF
FAAP24
MHF recruits FANCM to dsDNAHelicase ERCC4MHF
FANCM
Helicase MHFFANCM-N
rFANCM-N
MHF+ + + +_ ___
__
*
* MHF
* MHF
M-N
FANCM and MHF binds DNA synergistically
_ ___ _
_
+ + ++ rFANCM-N
MHF
*
*
M-NMHF
Fork
*
*
_ ___ _
_
+ + ++ rFANCM-N
MHF
HJ
M-NMHF
Most FANCM-MHF is present in a complex that is largely free of other FA core complex components
MHF1 IP
FANCM
MHF1MHF2
200
11697
66
55
36
31
21
14
6
HeLaNuclear Extract
MHF1 IP
A large percentage of FANCM-MHF is present in a complex independent of the FA core complex
MHF1 I
P
MHF and FANCM Form a DNA remodeling Complex that Protects Genome Stability from Human to Yeast
<30% of FANCM-MHF Most FANCM-MHF
Vertebrates Only All eukaryotes
Reconstituted FANCM-MHF has Higher Replication Fork Reversal Activity than FANCM alone
FANCM can catalyze fork regression
(b)
Reversal
b b
FANCMMHF
FANCM
FANCM + MHF
FANCM
bb
( Dr. Angelos Constantinou Lab )
MHF Is Required for stability and chromatin association of FANCM
MHF Is Required for Normal Monoubiquitination of FANCD2-FANCI and cellular resistance to MMC in human HeLa cells
MMC (ng/ml)
Surv
ival
(%)
0
0.2
0.4
0.6
0.8
1
0 100 200 300 400 500 600
siControl
siMHF2MHF2
FANCD2
Con
trol
Con
trol
MH
F2
MH
F2(siRNA)
FANCIC
ontr
ol
MH
F2
ACTIN
SL
LS
MMC Cisplatin
~1
MHF1
FANCM
5 15 30 45 min
FANCM and MHF Are Rapidly Recruited to DNA Interstrand Crosslinks induced by Laser-activated Psoralen
in S-phase cells
Mike Seidman’s group
S-phase HeLa cells+ laser-activated psoralen
- FANCM-MHF is recruited to replication forks blocked by ICLs
FANCM Recruitment to replication forks stalled by laser- induced ICLs is disrupted in MHF-depleted cells
siControl
siMHF2
siMHF1
γ-H2AX FANCM Merged
M. Seidman’s group
eCHIP—A new method to directly detect proteins recruited to DNA interstrand crosslinks in cells
(Lei Li’s group; Shen et al. Mol. Cell 2009)
MHF1 is recruited to DNA interstrand crosslinks; and its recruitment is stimulated by replication
Lei Li’s group
MHF and FANCM Work in the Same Pathway to Promote FANCD2 Monoubiquitination and to Suppress SCE in chicken DT40 cells
WT MHF1-/-
FANCM
-/-
MHF1-/-
FANCM
-/-
L/S: 1.54 0.34 0.16 0.16
DT40 cells:
MHF complex containing mutant A lacks DNA binding activity
AA AAmut B
MHF1
mut A
mut AB
RRK Rα1 α2 α3
Histone-fold
Mut
A
+ + + +
WT
Mut
AW
T
* MHF
*His-MHF210
20
50
25
120220
70
4030
WT Mut A
MHF1
MHF complex
MHF containing mutant A lacks DNA binding activity and fails to recruit FANCM to fork DNA
MHF
MHFFANCM-N
MHF
M-NMHF
The MHF1 mutant A has normal association with MHF2 and FANCM, whereas mutant B and AB have reduced association
MHF1 Requires its DNA binding activity to Promote normal FANCD2 Monoubiquitination
FANCM
MHF1
MHF2
BAF57
WT
MH
F1-/-
wt mut Amut Bmut A
B
FANCD2-LFANCD2-S
+ + + + + +
MHF1
MMC
L/S: 0.99 0.21 0.82 0.43 0.55 0.19
1 0.44 1.9 1.9 1.2 0.4
MHF1 Requires its DNA binding activity to efficiently
Suppress Sister-Chromatid Exchange
2.5
9.7
2.9
7.16.3
9.9
wt mut A mut B mut ABWT
MHF1-/-
4
0
8
12
16
20
24
28
Yeast MHF and FANCM Work in the Same Pathway to resist
MMS-induced cell killing
S. cerevisiae
MHF1
Mph1 (FANCM)
MHF2
( Dr. Kyungjae
Myung
Lab )
Cell numberuntreated 0.1% MMS
mhf1∆mhf2∆
mph1∆mph1∆mhf1∆mph1∆mhf2∆
wt
(srs2∆ strain)
The FANCM ortholog in S. Pombe is required for HR- dependent gene conversion at stalled replication forks
( Dr. Matthew C. Whitby Lab)
FANCM and MHF work in the same pathway to promote gene conversion at stalled replication forks in S. Pombe
( Dr. Matthew C. Whitby
Lab)
0 50 100 150 200Ade+ recombinant frequency (x10-4)
fml1∆
mhf2∆
wildtype
fml1∆
mhf2∆
conversion-types
MHF1
Fml1 (FANCM)
MHF2
A large portion of FANCM-MHF is present independent of the FA core complex; it may exist and protect genome stability in all eukaryotes
Summary• FANCM-MHF is a conserved DNA remodeling
complex required for:– Resistance to replication stress– Suppression of crossover recombination– Activation of FA-BRCA signaling pathway
• MHF is an essential partner of FANCM:– Provides a unique DNA binding surface– Promotes DNA binding and remodeling activity of
FANCM in vitro– Required for stability, chromatin-association, and
recruitment of FANCM to stalled forks in vivo
Yan et al. Mol. Cell in press; Meetei and Sung’s group, same issue
CoworkersNational Institute on Aging
Ruhikant MeeteiZhijiang YanChen LingDongyi Xu
Yin JinhuYutong XueYongjiang Li
Michael Seidman (LMG) Parameswary Muniandy
Ranjan Sen (LCMB)Hansen Du
University of LausanneAngelos ConstantinouAndrzej Stasiak
Cincinnati Children’s HospitalRuhikant Meetei
Cancer Research UKSteve WestAlberto Ciccia
VU University, NLHans JoenjeJohan de Winter
Oregon H&S Univ.Maureen Hoatlin
NHGRI/NIHKyungjae Myung
NCI/NIHYves Pommier
MD Anderson Cancer Center
Lei Li
University of WurzburgDetlev Schindler
University of OxfordIan Hickson
Matthew Whitby
University of MassachusettsChris Woodcock
University of TorontoJiongwen OuGrant Brown
Kawasaki Medicine SchoolMinoru Takata
Postdoct Positions Available: [email protected]
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