Nucleic
Acids
Research,
2023
1
https://doi.org/10.1093/nar/gkad624
FANCD2
and
RAD51
recombinase
directly
inhibit
DNA2
nuclease
at
stalled
replication
forks
and
FANCD2
acts
as
a
novel
RAD51
mediator
in
strand
exchange
to
promote
genome
stability
Wenpeng
Liu
1
,
5
,
†
,
Piotr
Polaczek
1
,
†
,
Ivan
Roubal
1
,
Yuan
Meng
2
,
5
,
Won-chae
Choe
1
,
Marie-Christine
Caron
3
,
Carl
A.
Sedgeman
1
,
Yu
Xi
5
,
Changwei
Liu
2
,
5
,
Qiong
Wu
2
,
Li
Zheng
2
,
Jean-Yves
Masson
3
,
4
,
Binghui
Shen
2
and
Judith
L.
Campbell
1
,
*
1
Br
aun
Labor
atories,
California
Institute
of
Technology,
Pasadena,
CA
91125,
USA,
2
Department
of
Cancer
Genetics
and
Epigenetics,
Beckman
Research
Institute,
City
of
Hope,
1500
East
Duarte
Road,
Duarte,
CA
91010-3000,
USA,
3
Genome
Stability
Laboratory,
CHU
de
Qu
́
ebec
Research
Center,
HDQ
Pavilion,
Oncology
Division,
9
McMahon,
Qu
́
ebec
City,
QC
G1R
3S3,
Canada,
4
Department
of
Molecular
Biology,
Medical
Biochemistry
and
Pathology;
Laval
University
Cancer
Research
Center,
Qu
́
ebec
City,
QC
G1V
0A6,
Canada
and
5
Colleges
of
Life
Sciences,
Zhejiang
University,
Hangzhou,
Zhejiang
310027,
China
Received
September
12,
2022;
Revised
June
17,
2023;
Editorial
Decision
July
05,
2023;
Accepted
July
28,
2023
ABSTRACT
FANCD2
protein,
a
key
coordinator
and
effector
of
the
interstrand
crosslink
repair
pathway,
is
also
re-
quired
to
pre
vent
e
xcessive
nascent
strand
degra-
dation
at
h
ydr
oxyurea-induced
stalled
f
orks.
The
RAD51
recombinase
has
also
been
implicated
in
regulation
of
resection
at
stalled
replication
forks.
The
mechanistic
contributions
of
these
proteins
to
f
ork
pr
otection
are
not
well
understood.
Here,
we
used
purified
FANCD2
and
RAD51
to
study
how
each
protein
regulates
DNA
resection
at
stalled
forks.
We
characterized
three
mechanisms
of
FANCD2-
mediated
f
ork
pr
otection:
(1)
The
N-terminal
domain
of
FANCD2
inhibits
the
essential
DNA2
nuclease
ac-
tivity
by
directly
binding
to
DNA2
accounting
for
over-
resection
in
FANCD2
defective
cells.
(2)
Independent
of
dimerization
with
FANCI,
FANCD2
itself
stabilizes
RAD51
filaments
to
inhibit
multiple
nucleases,
in-
cluding
DNA2,
MRE11
and
EXO1.
(3)
Une
xpectedl
y,
we
uncovered
a
new
FANCD2
function:
by
stabiliz-
ing
RAD51
filaments,
FANCD2
acts
to
stimulate
the
strand
exchange
activity
of
RAD51.
Our
work
bio-
chemicall
y
e
xplains
non-canonical
mechanisms
by
which
FANCD2
and
RAD51
protect
stalled
forks.
We
propose
a
model
in
which
the
strand
exchange
ac-
tivity
of
FANCD2
pr
o
vides
a
simple
molecular
expla-
nation
for
genetic
interactions
between
FANCD2
and
BRCA2
in
the
FA
/
BRCA
fork
protection
pathway.
GRAPHICAL
ABSTRACT
INTRODUCTION
Successful
completion
of
DNA
replication
requires
the
inte-
gration
of
many
proteins
and
pathways
that
protect,
repair
and
/
or
r
estart
r
eplication
forks.
The
principles
underlying
how
these
pathways
interact
and
are
regulated
to
maximize
*
To
whom
correspondence
should
be
addressed.
Tel:
+1
626
395
6053;
Fax:
+1
626
395
6948;
Email:
jcampbel@caltech.edu
†
The
authors
wish
it
to
be
known
that,
in
their
opinion,
the
first
two
authors
should
be
regarded
as
Joint
First
Authors.
Pr
esent
addr
ess:
Wenpeng
Liu,
Department
of
Biochemistry,
Vanderbilt
Uni
v
ersity
School
of
Medicine,
613
Light
Hall,
2215
Garland
Avenue,
Nashville,
TN
37232,
USA.
C
The
Author(s)
2023.
Published
by
Oxford
University
Press
on
behalf
of
Nucleic
Acids
Research.
This
is
an
Open
Access
article
distributed
under
the
terms
of
the
Creati
v
e
Commons
Attribution
License
(http:
//
creati
v
ecommons.org
/
licenses
/
by
/
4.0
/
),
which
permits
unrestricted
reuse,
distribution,
and
reproduction
in
any
medium,
provided
the
original
work
is
properly
cited.
Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkad624/7234524 by California Institute of Technology user on 29 August 2023
2
Nucleic
Acids
Research,
2023
genome
stability
have
yet
to
be
determined.
Fanconi
anemia
is
a
rare
disease
of
bone
marrow
failure,
de
v
elopmental
ab-
normalities,
and
cancer
predisposition.
At
the
cellular
le
v
el
it
is
diagnosed
by
sensitivity
to
DNA
interstrand
crosslink
(ICL)-inducing
agents
and
genome
instability.
Fanconi
ane-
mia
is
a
multigenic
disease
defined
by
at
least
22
comple-
mentation
groups,
including
many
regulatory
components,
nucleol
ytic
activities,
and
homolo
gy
dir
ected
r
epair
(HDR)
genes.
The
component
genes
suggest
a
coherent
pathway
for
maintaining
genome
stability
during
DNA
replication
that
goes
beyond
ICL
repair
and
includes
the
response
to
many
additional
types
of
r
eplication
str
ess
(
1
,
2
).
The
multigenic
character
of
the
FA
pathway
lends
itself
to
a
comprehen-
si
v
e
genetic
and
biochemical
dissection
(
3–14
).
FANCD2
is
a
key
regulator
of
the
FA
pathway
and
the
focus
of
our
current
studies
(
5
,
15
).
During
canoni-
cal
r
eplication-coupled
r
epair
of
ICLs,
after
a
r
eplication
fork
encounters
an
ICL,
FANCD2
and
a
related
pro-
tein
FANCI,
are
phosphorylated
by
activated
ATR
ki-
nase.
A
F
ANCD2
/
F
ANCI
heterodimer
is
also
formed,
and
FANCD2
in
this
heterodimer,
but
not
free
FANCD2,
is
mono-ubiquitylated
by
the
FA
core
complex,
containing
nine
FA
proteins,
including
the
FANCL
E3
ligase
complex
and
se
v
eral
associated
proteins.
FANCD2-ubi
is
involv
ed
in
both
activation
of
repair
e
v
ents
and
also
is
dir
ectly
r
e-
quired
in
the
later
enzymatic
repair
steps
at
strand
breaks
(
5
,
16–20
).
The
role
of
ubiquitin
is
to
enforce
stable
bind-
ing
of
F
ANCD2
/
F
ANCI
to
DN
A,
specificall
y
by
clamping
F
ANCD2-ubi
/
F
ANCI
heterodimers
onto
DNA
for
DNA
repair
(
21–24
).
In
addition
to
its
role
in
ICL
repair,
FANCD2
is
also
involved
in
the
recovery
of
stalled
replication
forks,
irre-
specti
v
e
of
the
source
of
DNA
damage
causing
replica-
tion
stress
(
6
,
7
,
11
,
14
).
Se
v
eral
studies
implied
that
non-
ubiquitylatable
F
ANCD2
(F
ANCD2-K561R)
could
not
re-
store
fork
protection
to
patient-deri
v
ed
FANCD2-defecti
v
e
cells
(
7
,
25
),
Other
results,
howe
v
er,
support
that
FANCD2
is
likely
to
have
constitutive
functions,
at
least
for
low
levels
of
r
eplication
str
ess,
such
as
endogenous
str
ess
(
1
,
2
).
With
respect
to
ubiquitylation,
the
study
of
FANCD2
knock-
out
and
knock-in
cell
lines
showed
that
cells
expressing
only
non-ubiquitylatable
FANCD2-K561R
had
much
less
se
v
ere
phenotypes
than
cells
with
a
FANCD2
knockout
(
26
).
Complementary
studies
showed
that
mutants
defecti
v
e
in
the
trans-acting
FA
core
complex
components
respon-
sible
for
ubiquitylation
of
FANCD2
are
less
sensiti
v
e
to
replication
fork
stalling
agents
than
FANCD2
knockdowns
or
knockouts
(
27
).
Importantly,
one
of
us
reported
that
FANCD2
can
protect
the
stalled
forks
by
different
mech-
anisms
than
FANCA
/
C
/
G,
members
of
the
core
complex
(
28
).
FANCD2
has
been
shown
to
interact
with
RAD51,
a
key
player
/
regulator
in
fork
protection,
and
to
do
so
in
a
ubiquitylation-independent
but
HU-stimulated
manner
(
29
).
At
a
stalled
fork,
induced
CMG
disassembly
disasso-
ciates
FANCD2
and
FANCI,
which
leads
to
fork
instabil-
ity
(
30
).
FANCD2
also
has
FANCI
independent
functions
(
11
,
27
,
31
).
FANCD2
deficient
cells
are
HU
and
aphidi-
colin
(a
DN
A
pol
ymerase
inhibitor)
sensiti
v
e,
while
FANCI
cells
are
not
(
27
).
These
results
stimulated
our
interest
in
studies
of
ubiquitin-
and
FANCI-
independent
roles
of
FANCD2.
Fork
protection,
operationally,
implies
protection
from
nucleases.
Se
v
eral
nucleases
hav
e
been
implicated
in
nascent
DNA
degrada
tion
a
t
DNA
structures
arising
a
t
stalled
forks
(
6
,
7
,
28
,
32
,
33
).
DNA2
helicase
/
nuclease
is
of
particular
in-
terest
because
it
is
essential
for
replication
in
normal
yeast
cells,
and
in
metazoans,
it
is
essential
for
normal
embryonic
de
v
elopment
(
34–36
).
Why
DNA2
is
essential
has
remained
a
matter
of
de
bate,
how
e
v
er.
While
synthetic
lethality
with
FEN1
deficiency
in
yeast
and
biochemical
characterization
suggests
that
DNA2
might
function
in
FEN1-independent
Okazaki
5
flap
r
emoval
(
35
,
36
),
mor
e
r
ecent
studies
show
that
DNA2
has
additional
important
functions,
raising
the
question
of
which
really
makes
it
essential.
DNA2’s
abil-
ity
to
remove
long
5
(or
3
)
ssDN
A
fla
ps
could
be
used
during
non-canonical
Okazaki
fragment
processing
in
the
presence
of
Pif1
(
37
,
38
),
also
see
(Hill
et
al.,
2020,
unpub-
lished
in
Biorixv).
It
could
also
pr
omote
contr
olled
resec-
tion
during
replication
fork
stalling
for
replication
fork
pro-
tection,
to
pre
v
ent
the
accumulation
of
aberrant
re
v
ersed
fork
intermediates
or
gaps
and
for
efficient
replication
fork
restart
(
32
,
39
,
40
).
DNA2
is
thought
to
be
especially
impor-
tant
a
t
dif
ficult-to-replica
te
sequences,
such
as
the
rDNA
(
41–43
),
telomeres
(
44–46
),
and
centromeres
(
47
).
Multi-
tasking
DNA2
is
also
involved
In
DNA
repair.
DNA2
per-
forms
long-range
resection
of
DSBs
during
homologous
re-
combination
(
48
),
in
conjunction
with
MRE11,
to
provide
3
ends
for
BRCA2-mediated
RAD51
filament
formation
and
strand
invasion.
We
discovered
that
DNA2-deficient
cells
are
sensiti
v
e
to
inter-
or
intr
a-str
and
crosslinks
induced
by
cisplatin
or
formaldehyde.
Paradoxically,
the
depletion
of
DNA2
in
cells
deficient
in
FANCD2
rescued
ICL
sen-
sitivity
in
FANCD2
mutants,
in
keeping
with
DNA2
be-
coming
toxic
in
the
absence
of
FANCD2
fork
protection
(
49
,
50
).
Se
v
eral
studies
confirm
tha
t
DNA2-media
ted
over-
resection
of
nascent
DNA
occurs
at
a
stalled
replication
fork
when
FANCD2
is
absent
(
26
,
28
,
30
,
32
,
33
,
36
,
51–54
),
suggesting
that
controlled
resection
by
DNA2
at
forks
is
es-
sential
for
replication
and
repair,
and
to
preserve
genome
stability.
The
question
remains
as
to
how
DNA2
is
precisely
controlled
at
replication
forks.
Answering
this
question
is
essential
to
understanding
how
both
FANCD2
and
DNA2
are
involved
in
fork
protection
and
maintenance
of
genome
stability
and
thus
understanding
their
roles
in
cancer
de
v
el-
opment
and
treatment.
RAD51
depletion
can
also
be
inhibitory
to
DNA2-
media
ted
degrada
tion
of
nascent
DNA
in
vivo
(
32
),
since
RAD51
has
been
shown
recently
to
promote
fork
reversal
using
its
recombination
activity
(
30
).
Furthermore,
a
domi-
nant
negati
v
e
RAD51
mutant
leads
to
e
xcessi
v
e
degrada-
tion
of
nascent
DNA
in
a
RAD51
T131P
/
WT
heterozy-
gote
and
this
over-resection
is
pre
v
ented
by
depletion
of
DNA2
(
53
).
FANCD2
and
RAD51
are
epistatically
linked
in
fork
protection
(
7
).
In
vivo
,
however,
it
is
not
known
if
RAD51
and
FANCD2
act
independently
or
together
and
whether
both
are
required
to
promote
fork
re
v
ersal
and
in-
hibit
DNA2.
Addressing
the
role
of
RAD51
in
protection
from
degradation
is
difficult
because
of
the
fact
that
RAD51
is
r
equir
ed
f
or
f
or
k
re
v
ersal
in
all
pathways
identified
to
date
(
28
,
55
).
Recently,
se
v
eral
studies
have
suggested
that
FANCD2
and
BRCA2,
the
RAD51
mediator,
perform
parallel
or
Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkad624/7234524 by California Institute of Technology user on 29 August 2023
Nucleic
Acids
Research,
2023
3
compensatory
functions
in
fork
protection
and
fork
recov-
ery
after
the
collapse
(
25
,
56
,
57
).
Since
BRCA2
is
thought
to
stabilize
RAD51
filaments,
we
hypothesized
that
FANCD2
may
provide
a
backup
source
of
this
BRCA2
function
in
re-
sponse
to
replication
stress.
This
mechanism
is
supported
by
the
fact
that
FANCD2
alone
and
F
ANCD2
/
F
ANCI
heterodimers
interact
physically
with
RAD51
(
29
,
32
,
58–
60
).
F
ANCD2
/
F
ANCI
comple
xes
hav
e
been
shown
to
in-
crease
RAD51
le
v
els
on
DNA,
but
the
specific
contribu-
tion
of
FANCD2
itself
and
the
relationship
of
this
obser-
vation
to
suppression
of
BRCA2
−
/
−
defects
has
not
been
established.
In
this
work,
we
studied
the
mechanisms
by
which
FANCD2
and
RAD51
mediate
fork
protection.
Our
in
vivo
results
confirm
that
FANCD2
is
r
equir
ed
to
pro-
tect
stalled
replication
forks
from
DNA2-dependent
over-
resection
after
acute
stress.
We
identified
at
least
two
poten-
tial
mechanisms
by
which
FANCD2
protects
nascent
DNA
from
nucleolytic
resection
in
vitro
:
(1)
FANCD2
inhibits
DNA2
nuclease
activity
directly
and
(2)
FANCD2
stabi-
lizes
RAD51
ssDNA
filaments
which
pre
v
ent
nucleolytic
digestion
by
multiple
nucleases.
Surprisingly,
FANCD2,
promotes
RAD51-mediated
strand
e
xchange
acti
vity
by
stabilizing
RAD51
on
ssDNA.
The
ability
to
stimulate
strand
exchange
suggests
that
FANCD2,
like
BRCA2,
is
a
RAD51
mediator.
Since
the
strand
exchange
activity
is
re-
quired
for
fork
reversal,
our
work
suggested
that
FANCD2
may
also
be
involved
in
fork
reversal
at
a
stalled
fork,
like
BRCA2
(
28
,
30
).
This
provides
a
novel
mechanistic
ex-
planation
for
the
dependency
of
BRCA2
−
/
−
tumors
on
FANCD2,
and
the
suppression
of
BRCA1
/
2
−
/
−
pheno-
types
b
y
elev
ated
le
v
els
of
FANCD2
(
25
,
56
,
57
),
Thus,
our
results
add
a
major
new
dimension
to
how
FANCD2
defi-
ciency
leads
to
loss
of
fork
protection,
leading
to
genome
instability
(
7
).
MATERIALS
AND
METHODS
Reagents
and
materials
See
Supplementary
Table
S1
in
Supporting
Material.
Cell
culture
U2OS,
A549
and
PD20
and
PD20
with
FANCD2
comple-
mented
cells
were
cultured
in
DMEM
medium
with
10%
FBS.
Nuclear
fractionation
Cells
(1
×
10
6
)
were
harvested
and
washed
with
PBS,
then
lysed
on
ice
for
20
min
with
100
l
H150
buffer,
which
con-
tains
50
mM
HEPES
(pH7.4),
150
mM
NaCl,
10%
glycerol,
0.5%
NP-40
and
protease
inhibitor
cocktail
(Roche).
The
lysate
was
spun
for
10
min
at
5000g,
and
the
supernatant
is
the
cytoplasmic
fraction.
The
pellet
was
washed
two
times
with
H150
lysis
buffer,
and
the
supernatant
discarded.
The
pellet
is
the
nuclear
fraction.
The
pellet
was
resuspended
in
PBS
(20
l)
and
20
l
2
×
SDS
loading
buffer
and
boiled
for
western
blot.
Immunofluor
escence
f
or
native
Br
dU
staining
and
EdU
staining
BrdU
staining
was
carried
out
as
described
(
61
).
Briefly,
cells
(1
×
10
5
labeled
with
BrdU
and
EdU
as
described
in
the
legend
to
Supplementary
Figure
S5)
were
plated
on
cov-
erslips,
washed
with
PBS,
pre-extracted
with
ice
cold
0.5%
Triton-X100
for
4
min,
then
fixed
with
4%
paraformalde-
hyde
for
10
min,
permeabilized
with
0.1%
Triton-X100
for
2
min
and
then
washed
with
PBS
3
times.
Blocking
was
carried
out
with
1%
BSA
in
PBS
for
1
h.
A
1
ml
click
re-
action
containing
5
l
1
mM
Azide-488
(Invitrogen),
100
l
20
mg
/
ml
sodium
ascorbate,
20
ul
100
mM
CuSO
4
)
was
performed
to
detect
incorporated
EdU.
Then
FANCD2
an-
tibody
(1:200
in
blocking
buffer)
was
added
and
incubated
overnight
a
t
4
◦
C
.
For
BrdU
staining,
slides
were
incuba
ted
with
BrdU
and
FANCD2
primary
antibody
overnight
at
4
◦
C.
The
slides
were
washed
in
PBS
three
times
and
then
incubated
with
secondary
antibody
(1:200,
Alexa
Fluor
594
and
488
fr
om
Invitr
ogen)
for
1
h
at
room
temperature.
The
slides
were
washed
with
PBS
3
times
and
mounted
with
Prolong
Gold
AntiFade
Reagent
with
DAPI
(Invitrogen
P36941).
Plasmid
and
siRNA
transfection
A549
and
U2OS
cells
were
plated
the
day
before
transfec-
tion.
20
nM
siRNA
was
used
for
single
and
16
nM
for
each
siRNA
in
co-transfection.
Cells
was
transfected
with
Gen-
mute
and
labeled
as
indicated
72
hours
post-transfection.
DNA2
plasmid
transfection
was
described
previously
(
36
).
DNA
fiber
assay
DNA
fiber
spreading
and
staining
were
performed
as
pre-
viousl
y
described
(
36
).
Briefly,
1000
labeled
cells
(2
l,
500
cells
/
l)
on
slides
were
half
dried,
10
l
lysis
buffer
(0.5%
SDS,
200
mM
Tris–HCl
pH
7.4,
50
mM
EDTA)
was
added,
followed
by
incubation
for
6
min
at
room
temperature.
The
slide
was
tilted
to
15
degrees
to
allow
the
DNA
to
run
slowly
down
the
slide.
Slides
were
air
dried
for
at
least
40
min-
utes
and
fixed
for
2
min
in
3:1
methanol:
acetic
acid
in
a
coplin
jar.
Slides
were
dried
in
a
hood
for
20
min.
Slides
were
treated
with
2.5
M
HCl
for
70
min
for
dena
tura
tion
and
then
washed
with
PBS
3
times
and
blocked
with
10%
goat
serum
in
PBST
(0.1%
Triton-X100
in
PBS)
for
1
h.
Slides
were
in-
cubated
with
the
rat
anti-BrdU
and
mouse
anti-BrdU
an-
tibody,
1:100,
for
2
h,
washed
3
times
with
PBS,
and
then
incubated
with
secondary
antibody
(Goat
anti-Mouse
488
and
Goat
anti-Rat
594,
Invitrogen)
at
1:200.
Slides
were
im-
aged
with
immunofluorescence
microscopy
and
fiber
length
measured
by
Nikon
software.
Statistical
analyses
were
com-
pleted
using
Prism.
An
ANOVA
test
was
used
when
com-
paring
more
than
two
groups
followed
by
a
Dunnett
multi-
ple
comparison
post-test.
Neutral
COMET
assay
The
neutral
COMET
assays
were
performed
in
accordance
with
the
manufacturer’s
(Trevigen)
instructions.
Cells
were
trypsinized
and
washed,
then
palleted,
resuspended
with
low
melt
agarose,
then
dropped
on
the
slides.
After
cooling
Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkad624/7234524 by California Institute of Technology user on 29 August 2023
4
Nucleic
Acids
Research,
2023
down,
the
slides
were
incubated
in
cold
lysis
buffer
(Trevi-
gen)
for
1
h,
then
incubated
in
running
buffer
for
30
min,
and
then
subjected
to
electrophoresis
at
21
V
for
45
min.
Slides
were
then
immersed
in
precipita
tion
buf
fer
(Trevigen)
and
70%
ethanol
for
30
min,
respecti
v
ely.
Slides
were
dried
overnight
and
stained
with
SYBR
green
I
(Thermofisher).
Slides
were
imaged
with
fluorescence
microscope
with
FITC
channel.
Immunoprecipitation
For
FLAG
pulldown
assays
and
immunoprecipitation
as-
says,
293T
cells
were
transfected
with
or
without
RAD51
vector
(or
FLAG-DNA2
vector)
using
the
Polyjet
(Signa-
Gen
SL100688)
transfection
reagent.
24
h
after
transfec-
tion,
the
cells
were
incubated
with
or
without
2
mM
HU
for
3
h.
Cells
(1
×
10
7
)
were
collected
and
lysed
by
brief
sonica-
tion
and
incubation
in
the
immunoprecipitation
(IP)
buffer
H150
(50
mM
HEPES–KOH
(pH7.4),
150
mM
NaCl,
0.1%
NP40
and
10%
glycerol)
with
protein
inhibitor
cocktail
(Thermo
Fisher)
for
30
min.
After
centrifugation
(20
000g,
15
min,
4
◦
C),
the
supernatants
were
collected,
and
the
pro-
tein
concentration
determined.
Cell
lysate
(1
mg)
was
pre-
cleaned
with
10
l
Protein
A
/
G
beads
(Thermo
#88802)
for
1
h.
After
removing
beads,
the
lysate
was
incubated
with
2
g
(1
g
/
l)
anti-RAD51
(ab133534
Abcam)
or
anti-
FLAG
M2
magnetic
beads
for
FLAG
pulldown
(Sigma).
Then
10
l
Protein
A
/
G
magnetic
beads
were
added
and
incubated
overnight
at
4
◦
C.
The
beads
were
washed
three
times
with
the
IP
buffer
H150
and
boiled
in
1
×
SDS-PAGE
loading
buffer
directly.
The
DNA2
and
FANCD2
were
an-
alyzed
by
western
blot
analysis.
Oligonucleotides
Oligonucleotide
substrates
for
enzymatic
assays
were
la-
beled
at
the
5
end
with
32
P
using
polynucleotide
kinase.
The
sequences
are
listed
in
the
Supplementary
Table
S1.
For
DN
A2
assays,
single-stranded
DN
A
was
JYM945
(
62
).
The
forked
substrate
was
designated
87
FORK.
The
5
flap
sub-
strate
was
LU
5
FLAP.
The
3
flap
substrate
was
LU
3
.
The
re
v
ersed
for
k
with
b
lunt
ends
consisted
of
4
oligonu-
cleotides:
str
and
1,
str
and
2,
str
and
3
and
str
and
4
in
the
Supplementary
Table
S1
(
63
).
The
reversed
fork
with
5
overhang
consisted
of
strand1L,
strandFANCD2,
strand3,
and
strand4.
The
MRE11
nuclease
duplex
substrate
was
formed
by
an-
nealing
5
labeled
JYM945
to
JYM925
(
62
).
This
was
also
used
for
binding
of
RAD51
to
dsDNA.
For
the
EXO1
as-
say,
a
hairpin
with
a
3
overhang
was
used.
For
RAD51
binding,
JYM945
was
used.
For
RAD51
strand
exchange
assays
the
single-stranded
DNA
was
EX-
TJYM925:
The
60mer
duplex
was
formed
by
annealing
la-
beled
JM945
to
JM925
(see
MRE11
substrate).
Proteins
Recombinant
human
RAD51
was
from
Abcam
(ab81943)
and
tested
for
ATPase
,
strand
exchange
,
and
DNA
binding
.
RuvC
was
Abcam
(ab63828).
MRE11
was
the
gift
of
Tanya
Paull
(UT
Austin)
and
EXO1
(0.77
mg
/
ml)
was
a
gift
from
Paul
Modrich,
Duke
Uni
v
ersity.
Sources
of
FANCD2
and
DNA2
are
described
in
the
text
or
figure
legend
describing
the
experiments
in
which
they
were
used.
FANCD2-his
purification
from
E.
coli
Human
FANCD2
protein
was
purified
from
E.
coli
as
previ-
ously
described
(
64
).
The
FANCD2
vector
was
transformed
into
BL21(DE3)
CodonPlus
(Agilent
Technologies
230280)
cells.
Twenty
liters
of
transformed
cells
were
amplified
at
30
◦
C,
250
rpm.
FANCD2
protein
was
produced
by
adding
0.5
mM
IPTG
at
16
◦
C
for
18
hours,
when
the
cell
den-
sity
reached
an
OD
600
=
0.6.
The
E.
coli
cells
were
har-
vested
and
pelleted
and
lysed
in
Buffer
A
(50
mM
Tris–
HCl
PH8.0,
500
mM
NaCl,
5
mM
2-mercaptoethanol,
1
mM
phen
ylmethylsulf
on
yl
fluoride
(PMSF),
12
mM
imida-
zole,
and
10%
glycerol),
and
disrupted
by
sonication.
The
lysate
was
centrifuged
at
20
000g
at
4
◦
C;
the
supernatant
was
mixed
gently
by
the
batch
method
with
3ml
of
Ni-NTA
agarose
beads,
at
4
◦
C
for
1
h.
The
beads
were
packed
into
an
Econo-column,
and
were
washed
with
67
column
volumes
of
buffer
A.
The
His-tagged
FANCD2
were
eluted
with
a
20
column
volumes
linear
gradient
of
12–400
mM
imidazole
in
buffer
A.
The
peak
fractions
were
collected.
To
remove
His
tag
from
the
FANCD2
protein,
thrombin
protease
(2U
/
mg
GE
healthcare)
was
added,
and
the
sample
was
then
di-
alyzed
against
4L
of
buffer
B
(20
mM
Tris–HCl,
pH8.0,
200
mM
NaCl,
5
mM
2-mercaptoethanol,
10%
glycerol).
Afterward,
the
sample
was
passed
through
a
Q
Sepharose
Fast
Flow
(2.5
ml,
GE
Healthcare)
column.
The
resin
was
washed
with
60
column
volumes
of
buffer
B
containing
250
mM
NaCl.
Human
FANCD2
was
then
eluted
with
a
20-
column
volume
linear-gradient
of
250
mM-450
mM
NaCl
in
buffer
B.
The
peak
fractions
were
collected,
and
human
FANCD2
was
further
purified
by
gel
filtration
chromatog-
raphy
on
a
Super
de
x
200
column
(GE
Healthcare)
equili-
bra
ted
with
Buf
fer
B
containing
200
mM
NaCl.
The
pu-
rified
FANCD2
was
concentrated,
frozen
in
aliquots,
and
stored
a
t
–80
◦
C
.
The
concentra
tion
of
purified
FANCD2
was
determined
by
the
Bradford
method,
using
BSA
as
standard.
FLAG-DNA2
purification
from
mammalian
cells
The
FLAG-DNA2
expression
and
purification
procedure
was
as
described
previously
(
44
).
In
brief,
whole
cell
lysates
were
incubated
with
the
M2
FLAG
magnetic
beads
(Sigma)
for
at
least
6
h
in
cold
room.
After
e
xtensi
v
ely
washing
with
a
buffer
containing
50
mM
Tris–Cl
(pH
7.5)
and
500
mM
NaCl,
the
bound
proteins
were
eluted
with
3
×
FLAG
pep-
tide
(Sigma).
The
purity
of
DNA2
proteins
was
analyzed
by
4–15%
gradient
SDS–polyacrylamide
electrophoresis
(SDS–PAGE)
and
Coomassie
brilliant
blue
staining,
and
the
concentration
was
determined
by
comparison
to
BSA
after
Coomassie
blue
staining
of
SDS
gels.
Mapping
the
FANCD2
binding
domain
in
DNA2
Mutant
FLAG-DNA2
proteins
were
prepared
using
site-
directed
mutagenesis.
The
N-terminal
deletions
were
made
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Nucleic
Acids
Research,
2023
5
using
the
HiFi
DNA
cloning
kit
from
NEB
to
excise
por-
tions
of
the
N-terminus
of
the
gene,
while
C-terminal
dele-
tions
were
made
by
the
insertion
of
a
stop
codon
earlier
in
the
gene
construct.
Coimmunopr
ecipitations
wer
e
per-
formed
by
ov
ere
xpressing
the
DNA2
proteins
in
HEK-293T
cells
prior
to
making
cell
lysates.
FANCD2
was
added
to
the
lysates
to
a
final
concentration
of
2
nM
protein
to
ensure
measur
able
inter
action
with
DN
A2.
The
FANCD2:DN
A2
complex
was
pulled
down
using
a
FANCD2
antibody
at-
tached
to
magnetic
beads.
The
beads
were
washed
prior
to
eluting
the
samples
using
SDS
loading
buffer,
and
the
samples
were
analyzed
by
western
blot
using
a
3
×
FLAG
antibody.
Strand
e
x
change
assays
Single-stranded
DNA
(EXTJYM925)
was
preincubated
in
the
presence
of
RAD51
and
FANCD2
in
a
reaction
mix-
ture
containing
25
mM
TrisOAc
(pH
7.5),
2
mM
MgCl2,
2
mM
CaCl2,
2
mM
ATP,
1
mM
DTT
and
0.1
mg
/
ml
BSA
for
5
min
at
37 ̊C
for
filament
formation.
Following
pre-incubation,
dsDNA
(5
labeled
JYM945
annealed
to
JYM925)
with
the
labeled
strand
complementary
to
the
fil-
ament,
was
added
to
the
reaction
mixture
and
incubation
was
continued
for
an
additional
30
min
at
37 ̊C
for
strand
exchange.
Reactions
were
terminated
by
the
addition
of
pro-
teinase
K
and
SDS
to
0.5
mg
/
ml
and
0.25%
respecti
v
ely
and
incubated
for
10
min
at
37
◦
C.
1
l
of
Loading
Buffer
(2.5%
Ficoll-400,
10
mM
Tris–HCl,
pH
7.5,
and
0.0025%
xylene
cyanol)
was
added
and
samples
were
loaded
on
an
8%
na-
ti
v
e
gel
using
29:1
30%
acrylamide
solution.
Gels
were
run
at
100
V
(constant
voltage)
for
4
h.
For
strand
exchange
assays
that
used
the
3
overhang
DNA
(RJ-167
annealed
to
RJ-PHIX-42-1)
for
filament
for-
mation
during
preincubation,
5
labeled
dsDNA
(5
labeled
RJ-Oligo1
annealed
to
RJ-Oligo2)
was
used
as
its
respec-
ti
v
e
strand
e
xchange
target
during
the
30
min
incubation.
Similarly,
in
instances
using
5
overhang
DNA
(RJ-167
an-
nealed
to
RJ-PHIX-42–2)
to
generate
filaments,
5
labeled
dsDNA
(5
labeled
RJ-Oligo4
annealed
to
RJ-Oligo3)
was
used
as
its
double-stranded
target.
Biotin
pull-down
assays
for
RAD51
and
FANCD2
associa-
tion
with
overhang
DNA
The
protocol
was
adopted
from
Jensen
et
al
.
Briefly,
the
oligonucleotide
substrate
Bio-RJ-PHIX-42–1
composed
of
the
same
sequence
as
RJ-PHIX-42–1
but
containing
a
3
biotin
modification
was
obtained
from
IDT
(Integrated
DN
A
Technolo
gies)
and
PAGE
purified.
The
biotinylated
3
overhang
substrate
was
generated
by
annealing
Bio-RJ-
PHIX-42–1
to
oligonucleotide
RJ-167
at
a
1:1
molar
ra-
tio
in
STE
buffer.
Competitor
heterolo
gous
dsDN
A
was
similar
ly
gener
ated
by
annealing
PAGE
purified
oligonu-
cleotides
Oligo#90
and
Oligo#60.
For
pull-down,
RAD51
and
FANCD2
proteins
were
in-
cuba
ted
in
Buf
fer
S
(25
mM
TrisOAC
pH
7.5,
1
mM
MgCl
2
,
2
mM
ATP,
1
mM
DTT
and
0.1
g
/
l
BSA)
for
15
min
at
37 ̊C
followed
by
the
addition
of
3
overhang
DNA
(162
nt
RJ-167
annealed
to
42
nt
3
Bio-RJ-PHIX-42–1)
and
competitor
heterologous
dsDNA
(90mer,
Oligo
#90
/
Oligo
#60
oligonucleotides)
and
the
reaction
was
incubated
for
an
additional
5
min
at
37 ̊C.
Where
DNA
was
omitted,
TE
buffer
was
used
and
similarly,
respecti
v
e
proteins
stor-
a
ge
b
uffers
were
used
where
proteins
were
omitted.
DNA-
protein
complexes
were
captured
by
adding
the
reaction
mixtures
to
2.5
l
of
MagnaLink
Streptavidin
magnetic
beads
(Solulink)
pre-washed
by
excess
Buffer
S
supple-
mented
with
0.1%
Ipegal
CA-630
and
rotating
for
10
min
at
25 ̊C.
Bead
complexes
were
then
washed
with
excess
Buffer
S
supplemented
with
0.1%
Ipegal
CA-630.
Protein
was
then
eluted
by
re-suspending
in
15
l
of
2
×
protein
sample
buffer
and
heating
at
54 ̊C
for
4
min.
The
elution
fraction
was
then
loaded
into
a
Bis-Tris
protein
gel
for
western
analy-
sis.
Following
transfer,
the
membrane
was
cut
horizontally
at
the
70
kDa
marker
to
separately
probe
for
RAD51
and
FANCD2.
The
lower
half
was
probed
using
1:1000
diluted
-RAD51
(Abcam)
and
the
upper
half
using
1:1000
diluted
-FLAG
(ThermoFisher)
to
detect
FANCD2.
Anti-mouse
(LI-COR)
secondary
antibody
diluted
1:10
000
was
used
and
membranes
were
imaged
via
Odyssey
imaging
system.
Bands
were
quantified
using
ImageQuant
(Cytiva)
software.
Nuclease
and
DNA-dependent
ATPase
assays
DNA2
nuclease
assay.
FANCD2-His
or
FANCD2-His
diluent
was
incubated
in
DNA2
nuclease
reaction
mix
(50
mM
HEPES–KOH,
pH
7.5,
5
mM
MgCl
2,
2mM
DTT,
0.25
mg
/
ml
BSA)
for
30
min
at
4
◦
C.
DNA2,
preincubated
with
substrate
(87
fork,
1.5
nM
molecules)
for
5
min
on
ice,
was
added
and
the
reaction
was
incubated
for
30
min
at
37
◦
C.
See
Supplementary
Table
S1
for
substrate
sequences.
Fol-
lowing
incubation,
proteinase
K
and
SDS
were
added
to
1
mg
/
ml
and
0.5%,
respecti
v
ely,
and
incubation
continued
for
10
min
at
37
◦
C.
Denaturing
termination
dye
(2X:
95%
deionized
formamide,
10
mM
EDTA,
0.1%
bromophenol
blue
and
0.1%
xylene
cyanol)
was
added
and
the
mixture
boiled
for
5
min.
Samples
were
run
on
a
sequencing
gel
and
the
gel
analyzed
by
phosphor
imaging.
Product
formation
was
determined
by
dividing
the
product
band
by
the
total
DNA
in
each
lane.
We
calculate
inhibition
by
determining
the
%
product
and
normalizing
to
the
control
lane
with
re-
specti
v
e
nuclease
alone
and
no
FANCD2.
MRE11
nuclease
assay.
MRE11
reaction
mixtures
con-
tained
25
mM
MOPS
(pH
7.0),
60
mM
KCl,
0.2%
Tween
20,
2
mM
DTT
and
1
mM
MnCl
2
as
described
(
65
).
MRE11
and
blunt
dsDNA
60mer
substrate
(JYM925
/
JYM945
oligonucleotides)
were
incubated
together
on
ice
for
5
min
before
being
introduced
to
the
reaction
mixture
at
200
nM
and
1
nM,
respecti
v
ely,
and
incubated
for
30
min
at
37
◦
C.
Following
incubation,
r
eactions
wer
e
terminated
by
adding
proteinase
K
and
SDS
was
added
to
1
mg
/
ml
and
0.5%
re-
specti
v
ely
and
incubated
for
10
min
at
37
◦
C.
10
l
of
2X
ter-
mina
tion
d
ye
was
added
and
samples
boiled
for
5
min.
After
denaturing,
samples
were
run
on
a
12%
sequencing
gel
at
constant
60
W
and
the
gel
analyzed
by
phosphor
imaging.
EXO1
nuclease
assay.
Conditions
are
as
previously
de-
scribed
(
66
).
Reaction
mixtures
(10
l)
contained
20
mM
Tris–HCl,
pH
7.6,
0.75
mM
HEPES–KOH,
120
mM
KCl,
250
g
/
ml
BSA,
2
mM
ATP,
1
mM
glutathione,
2
mM
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