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Loélia Babin
Alterations of mtDNA usingmito-TALENs:
deciphering mtDNA stability
• Themostcommonaberrancyandmosttoxicisa4977base-pair(bp)deletionspanningnucleotides8483– 13,459inmyopathic syndromes
• wasfirstdetectedintwoneuromusculardiseases– Kearns-Sayresyndrome(KSS)andprogressiveexternalopthalmoplegia (PEO)syndrome
• Linktoprematureages?• Correlatewithcertaintypesofcancer
Kazachkova etal,2013
Mitochondrial deletions
13bp
13bp
Formation of the common deletion:Two Hypothesis
Forkstalling ormisrepair ofbreaks?
Replication of mtDNA
EMimages
2Dgels
Invitro:POLG,twinkle,mtSSB
DNA combing a new way for studying replication of mtDNA
(A.Phillips;A.Sfeir NYU)Superresolution microscopie
Group A Group BOriH OriL
CldU – IdU
mtDNA replication by strand displacement
(A.Phillips;A.Sfeir NYU)
Efficientfiring ofthetwo origins
Efficientfiring of2origins
HT1080
MEFs, C2C12 cells, including undifferentiated myoblasts and differentiated myotubes
Twinkle patient mutations lead to stalled forkin the vicinity of the Common Deletion
OriL
OriH
OriL
OriH
Heavy strand
OriLLight strand
CldU Ab
IdU Ab
mtDNA FISH
mtDNA FISH
merge
merge
mtDNA FISH probeCldU
mtDNA FISH probeCldU
mtDNA FISH
merge
IdU & CldU Ab
Stalled Fork
(A.Phillips;A.Sfeir NYU)
Twinkle+/+
CldU Ab + IdU Ab + mtDNA (FISH probe)
Stal
led
Fork
Clone #2
13/45 13/460/49
TwinkleY508C/Y508CTwinkle+/+TwinkleR374Q/+
12/780/68
Clone #1
Twinkle R374Q/+
dominantnegative linkermutation
Twinkle Y508C recessive helicasemutation
Formation of the common deletion:Two Hypothesis
Forkstalling ormisrepair ofbreaks?
Protein
MLS
Induction of Targeted Breaks to mitochondria
gRNA
?
gRNA
AATGGGGATCGAGCTTAGCGATGGCTGCTACGGCGIIIIIIIIIIIIIIITTACCCCTAG IIIIIIIIIIIIIIIIIIIIIGCCGC
CTCGAATCGCTACCGACGATCas9
Nuclease
DNABinding DNABinding
TALENZincFinger Nuclease
CRISPR/Cas9
Mito- RE:PstI, SmaI,ApaI..SrivastavaandMoraes,2001Bayona-Bafaluy etal.,2005Mito-ZFN:Minczuk etal2008Mito-TALEN:Bacman 2013
Cas9
ZFN
gRNA
Cas9ZFN
PNPase UndefinedTransporter?
RE
RE
from RNA of RNaseP
3’ from RNA MRP
gRNA
Adapted from Bacman andal,Methods inenzymology 2014
11
MitoTALENs are sent to mitochondria
vBases modification/ gaps: Base Excision Repair (BER)
vAdducts: Nucleotide Excision Repair (NER)
vMismatches:Mismatche Repair (MMR)
vDSBsNon-Homologous End Joining (NHEJ)
Homologous Recombination12
lig4none or few modification short homology (Micro)
deletions
lig3/1
Classical NHEJ Alt-NHEJ
DSB
DNA repairNucleus Mitochondria?
NHEJclassique
KU=Ku70/Ku80 DNA-Pks
Xrcc4/LIgaseIV
PARP/MRN/CtIP
MicrohomologiesLonguesdélétions
CDB
NHEJalternatif
13
Non Homologous End Joining
vBases modification/ gaps: Base Excision Repair (BER)
vAdducts: Nucleotide Excision Repair (NER)
vMismatches:Mismatche Repair (MMR)
vDSBsNon-Homologous End Joining (NHEJ)
Homologous Recombination
vBases modification/ gaps: Base Excision Repair (BER)
vAdducts: Nucleotide Excision Repair (NER)
vMismatches:Mismatche Repair (MMR)??(no MSH2)
vDSBsNon-Homologous End Joining (NHEJ)?
- Classical (no LIG4) -- MMEJ/alt NHEJ (microhomologies)? (LIG3, POLQ)
Homologous Recombination?
v mtDNA degradation (repair? Replication?)v Deletion induction? 14
lig4none or few modification short homology (Micro)
deletions
lig3/1
Classical NHEJ Alt-NHEJ
DSB
DNA repairNucleus Mitochondria?
Induction of the Common Deletion using genome editing
v
OriH
OriL
R
CD
CDout
CD3’
CD5’
d-Loop
F
CDmid
CDin
12S –
mtDNA4977–CD3’ CD5’–ve CDout CDind-Loop CD5*
–ve CD3’ CDmidCD5’
DSB Nick-H Nick-L DSB*
12S –
mtDNA4977–
(A.Millet;S.Dubois)
FokI
FokI
T
T
5’ 3’
5’3’
Linker
Demi-répétition
Répétition
BrinH
BrinL
FokI dead
FokI
FokI
T
T
5’ 3’
5’3’
Linker
Demi-répétition
Répétition
BrinH
BrinL
FokI dead
o Nickase-H
o Nickase-L
Induction of the Common Deletion: Genetic requirements
v Ligase 3 : Only ligase in mitochondria- mtDNA replication- repair (BER)- alt NHEJ
LIGASE?
HA –γ-tubulin –
LIG3F/–
– +
0
50
100
150
+ Cre–!Cre
LIG1F/– LIG3F/–
**
Ref Ref
MRE11
siRNA-AsiCtrl
siRNA-B
0
50
100
150
Ref
MGME1
**
Ref
CtIP
Ref
– ++CD5’(nuclease)Ctrl
LIG1 –
HA –γ-tubulin –
LIG1F/–
– +Cre – ++CD5’(nuclease)Ctrl
MRE11
MRE11
siRNA-AControl siRNA-B+CD5’(nuclease)
MGME1
MGME1
siRNA-AControl siRNA-B+CD5’(nuclease)
CtIP
CtIP
siRNA-AControl+CD5’(nuclease)
% m
tDNA
4977
(n
orm
alize
d to
tota
l mtD
NA)
% m
tDNA
4977
(n
orm
alize
d to
tota
l mtD
NA)
DSB
break
DNA
resection
repeat
annealing
DNA
ligation
3’
5’
H strandL strand
Repair hypothesis
mtDNA4977mtDNA
(L.Babin,A.Millet)
Induction of the Common Deletion: Genetic requirements
v MRE11 : - repair (alt-NHEJ)- homologous recombination- mitochondria ?
v CtIP - repair (alt-NHEJ)- homologous recombination- mitochondria ?
v ExoG : - repair (BER)
v Mgme1 : - replicationv - repair (BER)
LIGASE? NUCLEASE?
HA –γ-tubulin –
LIG3F/–
– +
0
50
100
150
+ Cre–!Cre
LIG1F/– LIG3F/–
**
Ref Ref
MRE11
siRNA-AsiCtrl
siRNA-B
0
50
100
150
Ref
MGME1
**
Ref
CtIP
Ref
– ++CD5’(nuclease)Ctrl
LIG1 –
HA –γ-tubulin –
LIG1F/–
– +Cre – ++CD5’(nuclease)Ctrl
MRE11
MRE11
siRNA-AControl siRNA-B+CD5’(nuclease)
MGME1
MGME1
siRNA-AControl siRNA-B+CD5’(nuclease)
CtIP
CtIP
siRNA-AControl+CD5’(nuclease)
% m
tDNA
4977
(n
orm
alize
d to
tota
l mtD
NA)
% m
tDNA
4977
(n
orm
alize
d to
tota
l mtD
NA)
(L.Babin,A.Millet)
%mtADN
4977
(normalisé
parlem
tADN
total)
SicontrôleSiARN-ASiARN-B
DSB
break
DNA
resection
repeat
annealing
DNA
ligation
3’
5’
H strandL strand
Repair hypothesis
mtDNA4977mtDNA
LIGASE? NUCLEASE?
HA –γ-tubulin –
LIG3F/–
– +
0
50
100
150
+ Cre–!Cre
LIG1F/– LIG3F/–
**
Ref Ref
MRE11
siRNA-AsiCtrl
siRNA-B
0
50
100
150
Ref
MGME1
**
Ref
CtIP
Ref
– ++CD5’(nuclease)Ctrl
LIG1 –
HA –γ-tubulin –
LIG1F/–
– +Cre – ++CD5’(nuclease)Ctrl
MRE11
MRE11
siRNA-AControl siRNA-B+CD5’(nuclease)
MGME1
MGME1
siRNA-AControl siRNA-B+CD5’(nuclease)
CtIP
CtIP
siRNA-AControl+CD5’(nuclease)
% m
tDNA
4977
(n
orm
alize
d to
tota
l mtD
NA)
% m
tDNA
4977
(n
orm
alize
d to
tota
l mtD
NA)
DSB
break
DNA
resection
repeat
annealing
DNA
ligation
3’
5’
H strandL strand
Repair hypothesis
mtDNA4977mtDNA
HOMOLOGOUSRECOMBINATION
RAD51 –
HA –γ-tubulin –
RAD51
siRNA-AControl siRNA-B
RAD510
50
100
150
% m
tDNA
4977
(n
orma
lized
to to
tal m
tDNA
)
siRNA-AsiCtrl
siRNA-BRef
+CD5’(nuclease)
(L.Babin,A.Millet)
%mtADN
4977
(normalisé
parlem
tADN
total)
SicontrôleSiARN-ASiARN-B
HA-
RAD52
Tubuline
contrôle SiARN-A
+CD5’(nuclease)RAD52
0%
50%
100%
150%
200%
SiARNcontrôle
SiARNRAD52
%m
tADN
4977
(normalisé
parlemtADN
total)
RAD52
v RAD51 : - repair (HR)- mitochondria (yeast) ?
v RAD52 : repair (HR)- mitochondria ?
Induction of the Common Deletion: Genetic requirements
0
50
100
150
TWINKLE SSB
** ** ** *
POLG DNA2
siRNA-AsiCtrl
siRNA-B
Ref Ref Ref Ref
% m
tDNA
4977
(n
orm
alize
d to
tota
l mtD
NA)
HA –γ-tubulin –
POLGsiRNA-AControl siRNA-B
DNA2siRNA-AControl siRNA-B
TWINKLEsiRNA-AControl
SSBsiRNA-AControl
0 91.6 % inhibition (Q-RT-PCR)
POLG DNA2 SSB
fork
stalling
fork
slippage
H strandL strand
3’
5’
5’5’
5’5’
mis-annealing
of repeats +
Replication hypothesis
mtDNA4977 mtDNAmtDNA
replication
(A.Millet)
Induction of the Common Deletion: Genetic requirements
vPIF-1 : - replication- G quadruplexes
%mtADN
4977
(normalisé
parlem
tADN
total)
PIF-1
SicontrôleSiARN-ASiARN-B
TCAGGGTTTGTTATAATTTTTTATTTTTATGGGCTTtggtgagggaggtAGGTGGTG4
CD5’
Re-evaluation of G-quadruplex propensity with G4.HunterAmina Bedrat1,2,†, Laurent Lacroix3,*,† and Jean-Louis Mergny1,2,*
PIF1 stalled replication -> more deletionOr G4 stabilization -> more opened structure
more annealing of repeats
G4
G4G4G4
Induction of the Common Deletion: Genetic requirements
A unique replication-dependent repair pathway at the core of the mitochondrial common deletion
fork
stalling
fork
slippage
H strandL strand
3’
5’
5’5’
5’5’
mis-annealingof repeats
resection
& ligation
DNA
break
+
Replication hypothesis
(i)
(ii)
mtDNA4977 mtDNAmtDNA
Phillips,Milletetal,MolCell 2017
A unique replication-dependent repair pathway at the core of the mitochondrial common deletion
fork
stalling
fork
slippage
H strandL strand
3’
5’
5’5’
5’5’
mis-annealingof repeats
resection
& ligation
DNA
break
+
Replication hypothesis
(i)
(ii)
mtDNA4977 mtDNAmtDNA
Peeva etal,NatureCom2018
mtDNA degradation (repair? Replication?)
mtDNA released from the mitochondrial matrix to the cytosol -> interacts with cGAS and triggers activation of an effective inflammatory response mediated by type I IFNs(Rongvaux et al., 2014; West et al., 2015; White et al., 2014)
Stenella
coeru
leoalb
a
Porphyra purpurea
Mus musculus
Naegleria gruberi
Myxine glutinosa
Spadella cephaloptera
Cho
loep
us h
offm
anni
Placopecten magellanicus
Asymm
etron inferumCiona intestinalis
Sagitta enflata
Equu
s cab
allus
feru
s
Sipunculus nudus
Microm
onas sp RC
C299
Canis lupus fa
miliaris
Tupaia belangeri
Propithecus verreauxi
Chlorocebus pygerythrus
Chaetopterus variopedatus
Paedocypris progeneticaCarcharodon carcharias
sillo
cive
rb a
giso
noM
Caeno
rhabd
itis el
egan
s
Plasmodium
mexicanum
Lepidodermella squamata
Tarsius syrichta
Sacc
haro
myc
es c
erev
isia
e S2
88c
Gal
lus
gallu
s
Pan troglodytes
Epiperipatus biolleyi
Phaeodactylum tricornutum
Elep
has m
axim
us
Phytophthora andina
Paramecium
aurelia
Strongylocentrotus purpuratus
Met
agon
imus
yoko
gawai
Callithrix jacchus
Spermophilus dauricus
Delphinus capensis
suci
nopa
j of
uB
Tric
hopl
ax a
dhae
rens
Gasterosteus w
heatlandi
Mne
mio
psis
leid
yi
Drosophila melanogasterFa
lco
pere
grin
us
Lineus viridis
Presbytis melalophosAp
hroc
allis
tes
vast
us
Kom
agat
aella
pha
ffiiTetrahym
ena thermophila
Aotus trivirgatus
Paracercomonas marina
Hirudo nipponia
Leishmania tarentolae Hypsibius d
ujardini
Dicero
s bico
rnis
Cro
cody
lus
nilo
ticus
ebmop secy
morahccasozihcS
Brachionus plicatilis
Oroperipatus sp DVL-2011
Loxo
dont
a af
rican
a
Bugula neritina
Ascaris
suum
Tribolium castaneum
Pongo pygmaeus
Salmo salar
Reclinomonas americana
Oryza sativa Indica G
roup
Halicry
ptus s
pinulo
sus
Vaccinia virus
Loxocorone allax
Dictyostelium discoideum
Terebratulina retusa
Cyanidioschyzon merolae
Sph
enod
on p
unct
atus
Gnath
osto
mula
par
adox
a
Flustra foliacea
Phaco
choe
rus af
rican
us
Chlam
ydomonas reinhardtii
Sebastes oblongus
Arabidopsis thaliana
Lampetra fluviatilis
Mytilus californianus
Saimiri boliviensis boliviensis
Kudo
a he
xapu
ncta
ta
Amph
imed
on q
ueen
slan
dica
Thulinius sp DVL-2010
Felis catus
Enterobacteria phage T7
Loxosomella aloxiata
Taen
ia so
lium
Macracanthorhynchus hirudinaceus
Can
dida
alb
ican
s S
C53
14
Panthera pardus
Callorhinchus m
ilii
Styela plicata
Centruroides limpidus
Urechis unicinctus
eawakihsi anarrod
O
Papio anubis
Trich
echu
s m
anat
us
Neophoca cinerea
Nectonemertes cf mirabilis HC-2011
Priapu
lus ca
udatu
s
Pleu
robr
achi
a ba
chei
Terebratalia transversa
Ory
cter
opus
afe
r
Rhabdopleura compacta
Phascolosoma esculenta
Gorilla gorilla gorilla
Physcomitrella patens
Branchiostoma floridae
Anisakis
simplex
Rattus norvegicus
Philodina citrina
Saccoglossus kowalevskii
Thalassiosira pseudonana
Pseu
dotra
pelu
s si
naitu
s
Stachyamoeba lipophora
Acipenser oxyrinchus
Panthera leo
Homo sapiens
Cucumaria miniata
Sus sc
rofa
domes
ticus
Orcinu
s orca
Aqui
la c
hrys
aeto
s
Rhodomonas salina
Chry
saor
a qu
inqu
ecirr
haCelleporella hyalina
Xenopus laevis
Pavo
cris
tatu
s
Martes martes
Lepus americanusAmphiporus formidabilis
Ampl
exid
iscus
fene
stra
fer
Urechis caupo
Elep
hant
ulus
sp
VB00
1
Danio rerio
Leptorhynchoides thecatus
Gna
thos
tom
ula
arm
ata
Vermamoeba vermiformis
Hemiselm
is andersenii
15
10 9 8 7 6 5 4 3 2 1 0
g
Viru
s
pre plants
FungiArchaeplastida
Invertebrates
Non mammalian vertebrates
mam
malian
non p
rimate
s
prim
ates
Choanoflagellate
# repeats/mtDNA *100
Founder event orselection pressure
TowardsreducedrepetitiveDNAinthemitochondrialgenomeofhighereukaryotes
33repeatsinhuman…>=13bp?
Phylogenetic analysis: Repeat size >=13 bp/ genome size
Dynamics of the Genomeand Immune System
ErikaBrunetLoélia BabinArmelMilletAnnaSolé FerréElisaYaniz Galende
Jean-PierredeVillartayAurélieBerlandBenoitRochSatish TadiStefania MusilliVincentAbramowski
PatrickRevyLaëtitiaKermassonBenoitFranceMarieDaCruzMouniaHerragDespina MoshousKarineEudesMartaBenavides Nieto
A.Millet
L.Babin
Loélia Babin
NewYorkUniversity,USAgnel SfeirAaronFillipsMarcoTigano
HumanForntierScienceProgram
MNHN-CGiovannangeli-JPConcordet-ADeCian
