1
CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity 1
2
Juan A. Cantres-Veleza, Justin L. Blaizea, David A. Vierraa, Rebecca A. 3
Boisverta, Jada M. Garzonb, Benjamin Pirainoa, Winnie Tanc, Andrew J. 4
Deansc, and Niall G. Howletta,* 5
6
aDepartment of Cell and Molecular Biology, University of Rhode Island, Kingston, 7
Rhode Island, U.S.A 8
bDepartment of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical 9
School, Boston, MA 10
cGenome Stability Unit, St. Vincent’s Institute of Medical Research, Fitzroy, VIC 3065, 11
Australia 12
13
*Corresponding author: Niall G. Howlett Ph.D., 379 Center for Biotechnology and Life 14
Sciences, 120 Flagg Road, Kingston, RI, USA, Tel.: +1 401 874 4306; Fax: +1 401 874 15
2065; Email address: [email protected] 16
17
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2
Keywords 18
Fanconi anemia, FANCD2, CDK phosphorylation, cell cycle, ubiquitination 19
20
Abstract 21
Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone 22
marrow failure and cancer. The FA proteins function together to repair damaged DNA. 23
A central step in the activation of the FA pathway is the monoubiquitination of the 24
FANCD2 and FANCI proteins under conditions of cellular stress and during S-phase of 25
the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in 26
particular, are poorly understood. In this study, we have identified a CDK regulatory 27
phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 28
S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 29
phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination 30
of FANCD2 during S-phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing 31
S592 mutants display reduced proliferation under conditions of replication stress and 32
increased mitotic aberrations, including micronuclei and multinucleated cells. Our 33
findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 34
protein that promotes mitotic fidelity. 35
36
37
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Author Summary 38
Fanconi anemia (FA) is a rare genetic disease characterized by high risk for bone 39
marrow failure and cancer. FA has strong genetic and biochemical links to hereditary 40
breast and ovarian cancer. The FA proteins function to repair DNA damage and to 41
maintain genome stability. The FANCD2 protein functions at a critical stage of the FA 42
pathway and its posttranslational modification is defective in >90% of FA patients. 43
However, the function, and regulation of FANCD2, particularly under unperturbed 44
cellular conditions, remains remarkably poorly characterized. In this study, we describe 45
a novel mechanism of regulation of the FANCD2 protein during S-phase of the cell 46
cycle. CDK-mediated phosphorylation of FANCD2 on S592 promotes the 47
ubiquitination of FANCD2 during S-phase. Disruption of this phospho-regulatory 48
mechanism results in compromised mitotic fidelity and an increase in mitotic 49
chromosome instability. 50
51
Introduction 52
Protection of the integrity of our genome depends on the concerted activities of 53
several DNA repair pathways that ensure the timely and accurate repair of damaged 54
DNA. These DNA repair pathways need to be tightly coordinated and regulated upon 55
cellular exposure to exogenous DNA damaging agents, as well as during the cell cycle. 56
Somatic disruption of the DNA damage response leads to mutation, genome instability 57
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and cancer. In addition, germline mutations in DNA repair genes are associated with 58
hereditary diseases characterized by increased cancer risk and other clinical 59
manifestations. Fanconi anemia (FA) is a rare genetic disease characterized by 60
congenital abnormalities, increased risk for bone marrow failure and cancer, and 61
accelerated aging [1]. FA is caused by germline mutations in any one of 23 genes. The 62
FA proteins function together in a pathway to repair damaged DNA and to maintain 63
genome stability. A major role for the FA pathway in the repair of DNA interstrand 64
crosslinks (ICLs) has been described [2]. 65
The main activating step of the FA pathway is the monoubiquitination of the 66
FANCD2-FANCI heterodimer (ID2), which is catalyzed by the FA-core complex, 67
comprising FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, 68
UBE2T/FANCT, and the FA associated proteins. Monoubiquitination occurs following 69
exposure to DNA damaging agents and during S-phase of the cell cycle [3, 4]. 70
Recently, it was discovered that the monoubiquitination of FANCD2 and FANCI leads 71
to the formation of a closed ID2 clamp that encircles DNA [5, 6]. Moreover, 72
ubiquitinated ID2 (ID2-Ub) assembles into nucleoprotein filament arrays on double-73
stranded DNA [7]. The monoubiquitination of FANCD2 is necessary for efficient 74
interstrand crosslink repair, the maintenance of common fragile site stability and faithful 75
chromosome segregation, events that are all crucial for genomic stability [8, 9]. 76
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A DNA damage-independent role for the FA pathway during the cell cycle has 77
been implicated in several studies. For example, FANCD2 promotes replication fork 78
protection during S-phase and ensures the timely and faithful replication of common 79
chromosome fragile sites (CFSs) [8, 10, 11]. FANCD2 has also been shown to be 80
involved in mitotic DNA synthesis (MiDAS) during prophase and is present on the 81
terminals of anaphase ultrafine bridges [12, 13]. During the cell cycle FANCD2 82
monoubiquitination is maximal during S-phase and minimal during M-phase [4]. 83
FANCD2 and FANCI are phosphorylated by the ATR and ATM kinases following 84
exposure to DNA damaging agents, promoting their monoubiquitination [14-17]. 85
However, in general, the function and regulation of FANCD2 and FANCI during the 86
cell cycle remains poorly understood. 87
Cyclin-dependent kinases (CDKs) play a major role in regulating cell cycle 88
progression, with CDK-mediated hyperphosphorylation of the retinoblastoma protein 89
(pRb) being the primary mechanism of cell cycle regulation. CDKs are also known to 90
regulate DNA repair during the cell cycle. Several protein components of the 91
homologous recombination repair (HR), translesion DNA synthesis (TLS), non-92
homologous end joining (NHEJ), and telomere maintenance pathways are CDK 93
substrates [18]. For example, the BRCA2 protein is phosphorylated by CDK1 as cell 94
progress towards mitosis. CDK-mediated phosphorylation of BRCA2 blocks interaction 95
with RAD51 and thereby restricts HR DNA repair during M-phase [19]. Conversely, 96
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CDK1/2 phosphorylate the EXO1 nuclease to promote DNA strand resection and HR 97
repair during S-phase [20]. 98
In this study we have examined the regulation of the FANCD2 protein by 99
phosphorylation, specifically during the cell cycle. We show that FANCD2 is 100
phosphorylated on S592, a putative CDK site, during S-phase but not during M-phase 101
and is phosphorylated by CDK2 in vitro. Mutation of S592 as well as CDK inhibition 102
disrupts S-phase FANCD2 monoubiquitination. Furthermore, we demonstrate that FA-103
D2 (FANCD2-/-) patient cells stably expressing FANCD2 mutated at S592 display 104
reduced growth under conditions of replication stress, an altered cell cycle profile, and 105
increased genomic instability manifested as increased micronuclei, nucleoplasmic 106
bridges, and aneuploidy. Our results uncover important mechanistic insight into the 107
regulation of a key DNA repair/genome maintenance pathway under unperturbed 108
cellular conditions. 109
110
Results 111
FANCD2 is phosphorylated under unperturbed conditions and during S-phase of the 112
cell cycle 113
To study the phosphorylation of the FANCD2 protein in the absence and presence of 114
DNA damaging agents, we performed a lambda-phosphatase assay with several cell 115
lines following incubation in the absence or presence of the DNA crosslinking agent 116
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mitomycin C (MMC). Notably, we observed a large increase in FANCD2 mobility 117
following incubation of lysates with lambda-phosphatase even in the absence of MMC 118
in all cell lines examined (Fig. 1A). These results suggest that FANCD2 is subject to 119
extensive phosphorylation even in the absence of an exogenous DNA damaging 120
agent. A similar change in protein mobility was not observed for FANCI (Fig. 1A). To 121
determine if FANCD2 is subject to phosphorylation during the cell cycle, we performed 122
a double-thymidine block experiment causing an early S-phase arrest and analyzed 123
phosphorylation at regular time points following release. We observed maximal 124
phosphorylation of FANCD2 during S-phase of the cell cycle, with much less 125
phosphorylation observed as cells progressed through G2/M-and G1-phases of the cell 126
cycle (Fig. 1B). Again, we observed no appreciable change in FANCI mobility under the 127
same conditions, suggesting that FANCI is not subject to the same levels of 128
phosphorylation as FANCD2 during the cell cycle (Fig. 1B). Similar findings were 129
observed with U2OS cells (Fig. S1A and B). We also synchronized cells in M-phase 130
using nocodazole and, upon release, again observed maximal levels of FANCD2 131
phosphorylation during S-phase (~15 h after release) (Fig. S1C and D). The FANCA 132
protein is a central component of the FA core complex, a multi-subunit ubiquitin ligase 133
that catalyzes the monoubiquitination of FANCD2 [3, 21]. To determine if FANCD2 134
phosphorylation was dependent on its monoubiquitination, we performed a lambda-135
phosphatase assay with asynchronous and early S-phase synchronized FA-A (FANCA-/-) 136
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and FANCA-complemented FA-A cells. S-phase FANCD2 phosphorylation was 137
observed in the absence of FANCA, albeit to a slightly lesser extent than in cells 138
FANCA-complemented FA-A cells (Fig. 1C). These results suggest that S-phase 139
phosphorylation is coupled to, but not dependent upon, the monoubiquitination of 140
FANCD2. 141
142
FANCD2 is a CDK substrate 143
Phosphorylation of FANCD2 during the cell cycle suggested a role for cyclin-144
dependent kinases (CDKs) in the regulation of FANCD2. CDKs phosphorylate serine or 145
threonine residues in the consensus sequence [S/T*]PX[K/R], and several DNA repair 146
proteins are known CDK substrates, including BRCA2, FANCJ, and CtIP [19, 22, 23]. 147
Both FANCD2 and FANCI have several putative CDK phosphorylation sites with 148
varying degrees of conservation (Fig. S2A and B). To begin to assess the role of CDKs 149
in the regulation of FANCD2, we performed a double-thymidine block in the absence 150
and presence of the CDK inhibitors purvalanol A and SNS-032. At the concentrations 151
tested, treatment with both inhibitors resulted in a significant reduction in the levels of 152
FANCD2 and FANCI monoubiquitination observed after double-thymidine arrest and a 153
modest reduction in FANCD2 phosphorylation (Fig. 2A). Reduced FANCD2 154
phosphorylation was also observed upon incubation with other CDK inhibitors and 155
upon short-term exposure to purvalanol A (Fig. S2C and D). To determine if FANCD2 is 156
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a CDK substrate, we immunoprecipitated FANCD2 from FA-D2 (FANCD2-/-) patient 157
cells stably expressing V5-tagged LacZ or FANCD2, and probed immune complexes 158
with a pan anti-pS/T-CDK antibody. An immune reactive band was detected in immune 159
complexes from cells expressing FANCD2 and not LacZ (Fig. 2B), suggesting that 160
FANCD2 is a CDK substrate. We also performed an in vitro CDK kinase assay with 161
CDK2-Cyclin A and full length FANCD2 purified from High Five insect cells [24]. CDK-162
mediated FANCD2 phosphorylation was already evident upon purification from insect 163
cells (Fig. 2C). Following dephosphorylation with lambda phosphatase, we observed a 164
concentration-dependent increase in FANCD2 phosphorylation upon incubation with 165
CDK2-Cyclin A. These results indicate that FANCD2 is a CDK substrate and suggest 166
that FANCD2 monoubiquitination may be regulated or coupled with CDK 167
phosphorylation. 168
169
FANCD2 is phosphorylated on S592 during S-phase of the cell cycle 170
To map the in vivo sites of FANCD2 phosphorylation, we immunoprecipitated 171
FANCD2 from asynchronous U2OS cells stably expressing 3xFLAG-FANCD2 under 172
stringent conditions, and performed phosphoproteomic analysis using LC-MS/MS (Fig. 173
3A and B). Under these conditions, we observed the phosphorylation of multiple sites 174
including the previously detected ATM/ATR phosphorylation sites S1401 and S1404 175
(Table 1) [17]. We also detected phosphorylation of the putative CDK site S592 (Table 176
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1). To analyze the phosphorylation of FANCD2 during the cell cycle, we synchronized 177
HeLa cells in M-phase using nocodazole, immunoprecipitated FANCD2 immediately 178
upon release and at 15 h post-release and again performed phosphoproteomic 179
analysis using LC-MS/MS (Fig. 3C-F). Under these conditions, we detected 180
phosphorylation of FANCD2 on S592 in S-phase and not during M-phase. To study 181
FANCD2 S592 phosphorylation more closely, we generated a S592 phospho-specific 182
antibody. We arrested cells in M-phase using nocodazole and analyzed FANCD2 S592 183
phosphorylation upon release from nocodazole block. A FANCD2 pS592 184
immunoreactive band was observed at 12 h (S-phase) following release (Fig. 3G), 185
consistent with the results of our LC-MS/MS phosphoproteomic analysis (Fig. 3F, Table 186
1). Taken together our results suggest that the phosphorylation of FANCD2 S592 might 187
play an important regulatory function during S-phase (Fig. 3F). 188
189
Mutation of S592 disrupts S-phase FANCD2 monoubiquitination 190
FANCD2 S592 is predicted to localize to a flexible loop proximal to K561, the site of 191
monoubiquitination, suggesting a potential regulatory function for S592 192
phosphorylation (Fig. 4A). To begin to characterize the role of S592 phosphorylation, 193
we generated phospho-dead (S592A) and phospho-mimetic (S592D) variants and 194
stably expressed these in FA-D2 (FANCD2-/-) patient-derived cells (Fig. 4B). Mutation of 195
S592 did not adversely impact the stability or overall structure of FANCD2 as 196
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evidenced by equal protein expression levels and intact monoubiquitination following 197
MMC exposure (Fig. S3A). Previous studies have shown that FANCD2 undergoes 198
monoubiquitination as cells progress through S-phase of the cell cycle [4]. To analyze 199
the effects of S592 mutation on S-phase FANCD2 monoubiquitination, cells were 200
subject to a double-thymidine block, and FANCD2 monoubiquitination was analyzed 201
upon release. Consistent with previous studies, we observed an increased in 202
monoubiquitination of wild-type FANCD2 as cells progressed through S-phase (Fig. 203
4C). In contrast, S-phase monoubiquitination was markedly attenuated for both 204
FANCD2-S592A and FANCD2-S592D (Fig. 4C). We also analyzed levels of the mitotic 205
marker H3 pS10 in these cells. Compared to cells expressing wild-type FANCD2, we 206
observed persistent levels of H3 pS10 in FA-D2 cells expressing empty vector and the 207
FANCD2-S592A mutant (Fig. 4C). We observed a similar phenotype of elevated H3 208
pS10 in HeLa FANCD2-/- cells generated by CRISPR-Cas9 gene editing (Fig. S3B-D) 209
[25]. In contrast to cells expressing empty vector and the FANCD2-S592A mutant, we 210
observed a more rapid disappearance of H3 pS10 in cells expressing FANCD2-S592D 211
(Fig. 4C). In addition, FACS analysis of FA-D2 patient cells expressing empty vector 212
and the S592 variants upon release from double-thymidine block indicated a higher 213
percentage of cells in G2/M at earlier time points, compared to cells expressing 214
FANCD2-WT (Fig. S3E). Taken together, these results demonstrate that mutation of 215
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FANCD2 S592 disrupts S-phase monoubiquitination and leads to altered G2-M cell 216
cycle progression. 217
218
Mutation of FANCD2 S592 leads to decreased proliferative capacity and increased 219
mitotic defects 220
To assess the functional impacts of mutation of FANCD2 S592, we monitored the 221
proliferation of FA-D2 patient cells stably expressing FANCD2-WT and the S592 222
variants in the presence of low concentrations of DNA damaging agents for prolonged 223
periods using the xCELLigence real time cell analysis system. The xCELLigence system 224
enables the analysis of cellular phenotypic changes by continuously monitoring 225
electrical impedance. Impedance measurements are displayed as the cell index, which 226
provides quantitative information about the biological status of the cells, including cell 227
number, cell viability, and cell morphology. Compared to cells expressing FANCD2-228
WT, cells expressing empty vector and the S592 variants exhibited reduced 229
proliferation when cultured in the presence of low concentrations of the DNA 230
polymerase inhibitor aphidicolin (APH) (Fig. 5A and S5A). In contrast, mutation of 231
FANCD2 S592 had no discernible impact on growth in the presence of low 232
concentrations of mitomycin C (MMC) (Fig. 5A and S5A). We also analyzed growth in 233
the presence of the CDK1 inhibitor RO3306. CDK1 inhibition resulted in reduced cell 234
proliferation in cells expressing FANCD2-WT and the S592A variant compared to cells 235
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expressing empty vector or the S592D variant (Fig. S5B and C). Next, we examined the 236
effects of mutation of S592 on levels of mitotic aberrations. Compared to FA-D2 cells 237
expressing wild-type FANCD2, we observed increased levels of micronuclei, 238
nucleoplasmic bridges, and bi- and multi-nucleated cells in FA-D2 cells expressing the 239
S592 variants (Fig. 5B). Elevated levels of mitotic aberrations were also observed in FA-240
D2 cells expressing FANCD2-K561R, which cannot be monoubiquitinated (Fig. 5B). 241
Taken together, these results indicate that S592 phosphorylation is functionally 242
necessary to ensure mitotic fidelity and chromosome stability under non-stressed 243
conditions. 244
245
Discussion 246
In this study, we have investigated the posttranslational regulation of the central FA 247
pathway protein FANCD2 largely under unperturbed conditions. The majority of 248
studies to date have focused on the posttranslational regulation of FANCD2 and 249
FANCI following exposure to DNA damaging agents [15, 16, 26]. Here, we have 250
observed that FANCD2 undergoes extensive phosphorylation during the cell cycle 251
and, in particular, during S-phase of the cell cycle. Notably, in contrast, FANCI - a 252
FANCD2 paralog - does not appear to be subject to the same degree of 253
phosphorylation during the cell cycle. We have also determined that FANCD2 is 254
phosphorylated on S592 by CDK2-Cyclin A during S-phase using several approaches, 255
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including phosphoproteomic analysis of FANCD2 immune complexes from 256
synchronized cell populations and immunoblotting with a S592 phospho-specific 257
antibody. Previous studies have shown that FANCD2 is monoubiquitinated as cells 258
traverse S-phase, and this has been shown to contribute to the protection of stalled 259
replication forks from degradation [4, 11]. Here we show that mutation of S592, or CDK 260
inhibition, abrogates S-phase monoubiquitination, strongly suggesting that S592 261
phosphorylation primes FANCD2 for ubiquitination during S-phase. Recent studies 262
have established that the FANCL RING E3 ubiquitin ligase allosterically alters the active 263
site of the UBE2T E2 ubiquitin-conjugating enzyme to promote site-specific 264
monoubiquitination of FANCD2 [27]. Specifically, FANCL binding to UBE2T exposes a 265
basic triad of the UBE2T active site, promoting favorable interactions with a conserved 266
acidic patch proximal to K561, the site of ubiquitination [27]. We speculate that S592 267
phosphorylation may augment interaction with the basic active site of UBE2T. 268
Alternatively, S592 phosphorylation may inhibit FANCD2 de-ubiquitination by USP1, in 269
a manner similar to that previously reported for the FANCI S/TQ cluster [15]. 270
Our studies further emphasize the critical nature of coordinated posttranslational 271
modification of FANCD2. Several studies have previously established intricate 272
dependent and independent relationships between FANCD2 monoubiquitination and 273
phosphorylation. For example, the ATM kinase phosphorylates FANCD2 on several 274
S/TQ motifs following exposure to ionizing radiation, e.g. S222, S1401, S1404, and 275
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S1418 [17] - phosphorylation of S1401 and S1404 were also detected under 276
unperturbed conditions in this study. While phosphorylation of S222 promotes the 277
establishment of the IR-inducible S-phase checkpoint, S222 phosphorylation and K561 278
monoubiquitination appear to function as independent events [17]. In contrast, 279
phosphorylation of FANCD2 on T691 and S717 by the ATR kinase is required for 280
efficient FANCD2 monoubiquitination [16, 28]. CHK1-mediated phosphorylation of 281
FANCD2 S331 also promotes DNA damage-inducible monoubiquitination and its 282
interaction with FANCD1/BRCA2 [29]. Conversely, CK2-mediated phosphorylation of 283
FANCD2 inhibits its monoubiquitination and ICL recruitment [30]. Our results suggest 284
that CDK-mediated phosphorylation of S592 primes FANCD2 for monoubiquitination 285
during S-phase in particular. It remains to be determined if other posttranslational 286
modifications, including phosphorylation at other sites, e.g. S1401 and S1404 detected 287
in this study, also contribute to the regulation of FANCD2 during the cell cycle. 288
Our work aligns with several previous studies describing the coordination of the 289
DNA damage response with cell cycle progression. For example, BRCA2 carboxy-290
terminal phosphorylation by CDK2 precludes the binding of RAD51, thereby 291
inactivating HR prior to the onset of mitosis [19]. In contrast, CDK1/2 phosphorylation 292
of EXO1 endonuclease positively regulates DNA strand resection and HR repair during 293
S-phase [20]. Similarly, CDK-mediated phosphorylation of CtIP allosterically stimulates 294
its phosphorylation by ATM, resulting in the promotion of strand resection and HR 295
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during S-phase [31]. While a role for FANCD2 in HR has been established [32], it 296
remains to be determined if CDK-mediated FANCD2 S592 phosphorylation promotes 297
HR or other related functions. For example, FANCD2 has been shown to have a DNA 298
repair-independent role in protecting stalled replication forks from MRE11-mediated 299
degradation [11]. We have also previously established an important role for FANCD2 in 300
the maintenance of CFS stability: In the absence of FANCD2, CFS replication forks stall 301
at a greater frequency, a likely consequence of persistent toxic R-loop structures, 302
leading to visible gaps and breaks on metaphase chromosomes [8, 10, 33, 34]. Under 303
conditions of replication stress, CFSs can remain unreplicated until mitosis where 304
replication can be completed through a process referred to as mitotic DNA synthesis 305
(MiDAS), a POLD3- and RAD52-dependent process that shares features with break-306
induced DNA replication (BIR) [35, 36]. FANCD2 has been shown to play a role in 307
MiDAS [12, 13]. Defects in - or an overreliance upon - this process are likely to lead to 308
chromosome missegregation and mitotic defects, as we have observed upon mutation 309
of S592. Increased mitotic aberrations have previously been observed in primary 310
murine FA pathway-deficient hematopoietic stem cells and FA-patient derived bone 311
marrow stromal cells [9]. Taken together, our results support a model whereby CDK-312
mediated S592 phosphorylation promotes efficient S-phase monoubiquitination, 313
ensuring the efficient and timely completion of CFS replication and mitotic 314
chromosome stability. A greater understanding of the function and regulation of the FA 315
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pathway under physiologically relevant non-stressed conditions will provide greater 316
insight into the pathophysiology of this disease and ultimately lead to improved 317
therapeutic options for FA patients. 318
319
Materials and Methods 320
Cell culture and generation of mutant cell lines 321
HeLa cervical carcinoma and U2OS osteosarcoma cells were grown in Dulbecco's 322
modified Eagle's medium (DMEM) supplemented with 10% v/v FBS, L-glutamine and 323
penicillin/streptomycin. HeLa FANCD2-/- cells generated by CRISPR-Cas9 were 324
generously provided by Martin Cohn at the University of Oxford [25]. 293FT viral 325
producer cells (Invitrogen) were cultured in DMEM containing 12% v/v FBS, 0.1 mM 326
non-essential amino acids (NEAA), 1% v/v L-glutamine, 1 mM sodium pyruvate and 1% 327
v/v penicillin/streptomycin. PD20 FA-D2 (FANCD2hy/-) patient cells were purchased 328
from Coriell Cell Repositories (Catalog ID GM16633). These cells harbor a maternally 329
inherited A-G change at nucleotide 376 that leads to the production of a severely 330
truncated protein, and a paternally inherited missense hypomorphic (hy) mutation 331
leading to a R1236H change. PD20 FA-D2 cells were stably infected with 332
pLenti6.2/V5-DEST (Invitrogen) harboring wild-type or mutant FANCD2 cDNAs. Stably 333
infected cells were grown in DMEM complete medium supplemented with 2 μg/ml 334
blasticidin. 335
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18
336
Immunoprecipitation 337
FA-D2 cells stably expressing LacZ or V5-tagged FANCD2, U2OS, and U2OS 3xFLAG 338
FANCD2 cells were lysed in Triton-X100 lysis buffer (50 mM Tris.HCl pH 7.5, 1% v/v 339
Triton X-100, 200 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, Protease inhibitors (Roche), 340
and 40 mM b-glycerophosphate) on ice for 15 min followed by sonication for 10 s at 341
10% amplitude using a Fisher Scientific Model 500 Ultrasonic Dismembrator. Anti-V5 342
or anti-FLAG-agarose were washed and blocked with NETN100 buffer (20 mM Tris-343
HCl pH 7.5, 0.1% NP-40, 100 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 344
protease inhibitors (Roche)) plus 1% BSA and the final wash and resuspension was 345
done in Triton-X100 lysis buffer. Lysates were incubated with agarose beads at 4°C for 346
2 h with nutating. Agarose beads were then washed in Triton-X100 lysis buffer and 347
boiled in 1× NuPAGE buffer (Invitrogen) and analyzed for the presence of proteins by 348
SDS-PAGE and immunoblotting or stained using Colloidal Blue Staining Kit 349
(Invitrogen) for mass spectrometry. 350
351
Cell cycle FANCD2 immunoprecipitation 352
S-phase and M-phase synchronized populations of HeLa cells were lysed in lysis buffer 353
(20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 400 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.1 354
mM DTT, 0.1% NP-40, 1 mM PMSF and complete protease inhibitors). 100 units of 355
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19
benzonase was added per 100 µL of lysis buffer. Protein G magnetic beads 356
(Dynabeads, Novex) were crosslinked with anti-FANCD2 (NB100-182, Novus 357
Biologicals) antibody. An equal volume of no salt buffer (20 mM HEPES, pH7.9, 1.5 mM 358
MgCl2, 0.2 mM KCl, 0.2 mM EDTA, 0.1mM DTT, 0.1% NP-40, plus complete protease 359
inhibitors) was added to 2 mg of whole cell lysate. Samples were resuspended in anti-360
FANCD2-bound beads rotating at 4°C for 4 h. Beads were washed in wash buffer (20 361
mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM KCl, 300 mM KCl, 10% glycerol, 0.2 mM 362
EDTA, 0.1 mM DTT, 0.1% NP-40, plus complete protease inhibitor). The samples were 363
eluted in urea elution buffer (8 M urea, 1 mM Na3VO4, 2.5 mM Na₂H₂P₂O, 1 mM b-364
glycerophosphate and 25 mMHEPES, pH 8.0). Silver staining was performed to 365
visualize the IP and FANCD2 pulldown was confirmed by western blot. Samples were 366
sent for LC-MS/MS analysis at the COBRE Center for Cancer Research Development 367
Proteomics Core at Rhode Island Hospital. 368
369
Lambda phosphatase assay 370
Cells were harvested and pellets were split into two, lysed in either lambda 371
phosphatase lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 mM EGTA, 1 mM 372
dithiothreitol, 2 mM MnCl2, 0.01% Brij35, 0.5% NP-40, and protease inhibitor) or 373
lambda phosphatase buffer with the addition of phosphatase inhibitors, 2 mM Na3VO4 374
and 5 mM NaF for 15 min at 4°C followed by sonication for 10 s at 10% amplitude 375
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20
using a Fisher Scientific Model 500 Ultrasonic Dismembrator. Lysates were incubated 376
with or without 30 U of lambda phosphatase 1 h at 30°C. 377
378
Immunoblotting 379
For immunoblotting analysis, cell pellets were washed in PBS and lysed in 2% w/v 380
SDS, 50 mM Tris-HCl, 10 mM EDTA followed by sonication for 10 s at 10% amplitude. 381
Proteins were resolved on NuPage 3-8% w/v Tris-Acetate or 4-12% w/v Bis-Tris gels 382
(Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes. The 383
following antibodies were used: rabbit polyclonal antisera against CHK1 (2345, Cell 384
Signaling), CHK1 pS345 (2345, Cell Signaling), cyclin A (SC751, Santa Cruz), CDC2 385
pY15 (4539,Cell Signaling), FANCD2 (NB100-182; Novus Biologicals), FANCI (A301-386
254A, Bethyl), FLAG (F7425, Sigma), H3 pS10 (9701, Cell Signaling), pan pS/T-CDK 387
(9477, Cell Signaling), and V5 (13202, Cell Signaling), and mouse monoclonal antisera 388
against α-tubulin (MS-581-PO, Neomarkers). 389
390
Plasmids 391
The FANCD2 S592A and S592D cDNAs were generated by site-directed mutagenesis 392
of the wild type FANCD2 cDNA using the Quikchange Site-directed Mutagenesis Kit 393
(Stratagene). The forward (FP) and reverse (RP) oligonucleotide sequences used are as 394
follows: S592A FP 5′- CGGCAGACAGAAGTGAAGCACCTAGTTTGACCCAAG-3′; 395
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21
S592A RP 5′-CTTGGGTCAAACTAGGTGCTTCACTTCTGTCTGCCG-3’; S592D FP 5’-396
GGCGGCAGACAGAAGTGAAGATCCTAGTTTGACCCAAGAG-3’; and S592D RP 5’-397
CTCTTGGGTCAAACTAGGATCTTCACTTCTGTCTGCCGCC-3’. The full length 398
FANCD2 cDNA sequences were TOPO cloned into the pENTR/D-TOPO (Invitrogen) 399
entry vector, and subsequently recombined into the pLenti6.2/V5-DEST (Invitrogen) 400
destination vector and used to generate lentivirus for the generation of stable cell 401
lines. 402
403
Immunofluoresence microscopy 404
Hela or Hela FANCD2-/- generated by CRISPR-Cas9 were seeded in at a density of 2 x 405
104 in chamber slides (Millicell EZ Slide, Millipore) overnight. Cells were treated with 406
100 nM MMC for 24 h and fixed in fixing buffer (4% w/v paraformaldehyde, 2% w/v 407
sucrose in PBS, pH 7.4) at 4°C for 10 min. Cells were permeabilized using 0.3% v/v 408
Triton-X in PBS for 10 min at room temperature. Cells were blocked in antibody 409
dilution buffer (ADB) (5% v/v goat serum, 0.1% v/v NP-40 in PBS, pH 7.4) and then 410
incubated with mouse-anti H3 pS10 (9706, Cell Signaling) and rabbit-anti-FANCD2. 411
Cells were washed with PBS and incubated in secondary goat-anti-mouse Alexa Fluor 412
594 and donkey-anti-rabbit Alexa Fluor 488 in ABD for 1 h. Cells were washed in PBS 413
and stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Vector 414
Laboratories). 415
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22
416
Cell-cycle synchronization 417
For early S-phase arrest, cells were synchronized by the double-thymidine block 418
method. Cells were seeded in 10 cm2 dishes and treated with 2 mM thymidine 419
(226740050, Acros Organics) for 18 h. Cells were washed with PBS and released into 420
thymidine-free media for 10 h following by a second incubation in 2 mM thymidine for 421
18 h. Cells were washed twice with phosphate-buffered saline (PBS) and released into 422
thymidine-free media. For M-phase arrest, cells were synchronized using the mitotic 423
shake-off method. HeLa cells were seeded in 10 cm2 dishes. Cells were treated with 424
100 ng/mL of nocodazole (SML1665, Sigma) at 80-90% confluency for 15 h. Mitotic 425
cells were collected by the shake off method and re-plated in nocodazole-free media 426
for cell cycle progression analysis. For late G2-phase arrest, cells were synchronized by 427
reversible inhibition of CDK1 (Vassilev 2006). Cells were seeded in 10 cm2 dishes and 428
treated with 6 µM R03306 (15149, Cayman Chemicals) for 16 h. Cells were washed 429
with PBS and released into fresh media without R03306. 430
431
Cell cycle analysis by FACS 432
Cells were fixed in ice cold methanol, washed in PBS and incubated in 50 μg/mL 433
propidium iodide (PI) (Sigma) and 30 U/mL RNase A for 10 min at 37°C, followed by 434
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23
analysis using a BD FACSVerse flow cytometer. The percentages of cells in G1, S, and 435
G2/M were determined by analyzing PI histograms with FlowJo V10.2 software. 436
437
In-vitro CDK phosphorylation assay 438
Purified FANCD2 CDK2-Cyclin A proteins were a generous gift from Andrew Deans at 439
the University of Melbourne. In order to remove any previous phosphorylation 2 µg of 440
protein were incubated with 100 U of lambda phosphatase, 10 mM protein 441
metallophosphatases (PMP) and 10 mM MnCl2 at 30oC for 30 min. For the 442
phosphorylation reaction, each reaction tube was composed of the following reagents 443
in this specific order: 10x CDK kinase buffer (250 mM Tris pH 7.5, 250 mM 444
glycerophosphate, 50 mM EGTA, 100 mM MgCl2), 2 µg of protein, 15 nM 445
CDK2:Cyclin A, and 20 µM ATP. Samples were incubated at 30oC for 30 min and the 446
reaction was stopped by adding 10 µL of 10% b-mercaptoethanol in 4x LDS buffer. 447
448
Cell proliferation assay 449
For cell proliferation assays we performed electrical impedance analysis using the 450
xCELLigence RTCA DP system from Acea Biosciences. Cells were seeded in 451
polyethylene terephthalate (PET) E-plates (300600890, Acea Biosciences). Cells were 452
incubated in the absence or presence of 0.4 µM aphidicolin (APH) or 20 nM mitomycin 453
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24
C (MMC) for 120 h. Electrical impedance measurements were taken every 15 min over 454
the course of the incubation. Statistical analysis was performed using R. 455
456
Acknowledgements 457
We thank members of the Howlett, Camberg, and Dutta laboratories for critical 458
discussions. We thank Andrew A. Deans and Winnie Tan at the University of Melbourne 459
for purified proteins and Martin A. Cohn at the University of Oxford for HeLa FANCD2-/- 460
cells. This work was supported by NIH/NIGMS grant R01HL149907 (N.G.H.), an 461
American Society of Hematology Bridge Grant (N.G.H.); Rhode Island IDeA Network of 462
Biomedical Research Excellence (RI-INBRE) grant P20GM103430 from the National 463
Institute of General Medical Sciences; and Rhode Island Experimental Program to 464
Stimulate Competitive Research (RI-EPSCoR) grant #1004057 from the National 465
Science Foundation. We declare that we have no conflicts of interest. 466
467
468
469
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25
Figure Legends 470
Figure 1. FANCD2 is phosphorylated under non-stressed conditions and during S-471
phase of the cell cycle 472
(A) HeLa, U2OS and COS-7 cells were incubated with or without 200 nM MMC for 24 473
h. Cells were harvested and lysed in lambda phosphatase lysis buffer, incubated in the 474
absence or presence of lambda phosphatase, and the indicated proteins were 475
analyzed by immunoblotting. (B) HeLa cells were synchronized at the G1/S boundary 476
by a double-thymidine block and released into thymidine-free media. Cells were lysed 477
in lambda phosphatase buffer, incubated in the presence or absence of lambda 478
phosphatase, and lysates analyzed by immunoblotting. For cell cycle stage analysis, 479
cells were fixed, stained with propidium iodide and analyzed by flow cytometry. (C) 480
FA-A (FANCA-/-) and FANCA-complemented FA-A cells were synchronized at the G1/S 481
boundary by a double-thymidine block and released into thymidine-free media. Cells 482
were harvested, lysed in lambda phosphatase buffer, incubated in the presence or 483
absence of lambda phosphatase, and analyzed by immunoblotting. 484
485
Figure S1. Phosphorylation of FANCD2 during S-phase of the cell cycle 486
(A) U2OS cells were synchronized at the G1/S boundary by a double-thymidine block 487
and released into thymidine-free media. Cells were harvested, lysed in lambda 488
phosphatase buffer and incubated in the presence or absence of lambda 489
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26
phosphatase. Samples were analyzed by immunoblotting. (B) Cells were fixed, stained 490
with propidium iodide and analyzed by flow cytometry to determine cell cycle stage. 491
(C) HeLa cells were synchronized in M-phase with a nocodazole block. Mitotic cells 492
were physically detached by the shake-off method and released into nocodazole-free 493
media. Cells were harvested, lysed in lambda phosphatase buffer and incubated in the 494
presence or absence of lambda phosphatase. Samples were analyzed by 495
immunoblotting. (D) Cells were fixed, stained with propidium iodide and analyzed by 496
flow cytometry to determine cell cycle stage. 497
498
Figure 2. FANCD2 is a CDK substrate 499
(A) HeLa cells were synchronized at the G1/S boundary by a double-thymidine block 500
performed in the absence or presence of the CDK inhibitors Purvalanol A (PURV-A) or 501
SNS-032. Cells were lysed in lambda phosphatase buffer, incubated in the absence or 502
presence of lambda phosphatase, and analyzed by immunoblotting. Cells were also 503
fixed, stained with propidium iodide and analyzed by flow cytometry to determine cell 504
cycle stage. (B) FA-D2 (FANCD2-/-) cells stably expressing LacZ-V5 or FANCD2-V5 505
were immunoprecipitated with anti-V5 agarose and immune complexes were 506
immunoblotted with anti-FANCD2 and anti-pS-CDK antibodies. (C) Purified FANCD2 507
was incubated in the absence (-) or presence (+) of lambda phosphatase followed by 508
incubation with increasing concentrations of CDK2-Cyclin A and ATP at 30°C for 30 509
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min. Samples were immunoblotted with a pan anti-pS-CDK antibody or stained with 510
SimplyBlue SafeStain. 511
512
Figure S2. FANCD2 is a CDK substrate 513
(A-B) Shown is an alignment of mouse, human, and chicken FANCD2 (A) and FANCI 514
(B) amino acid sequences using the T-Coffee server, with the secondary structure of 515
mouse FANCD2 or FANCI illustrated. Putative CDK phosphorylation sites are shaded 516
in yellow. (C) HeLa cells were incubated in the absence (NT) or presence of 10 μM 517
olumucine (Olo), 10 μM olomucine II (Olo II), 10 μM purvalanol A (Pur), 10 μM RO3306 518
(Ro) and 20 μM roscovitine (Rosc) for 24 h. Whole cell lysates were incubated in the 519
absence or presence of lambda phosphatase and analyzed by immunoblotting. (D) 520
HeLa cells were treated with 10 μM purvalanol A for the indicated times, lysates were 521
incubated in the absence or presence of lambda phosphatase, and analyzed by 522
immunoblotting. 523
524
Figure 3. FANCD2 is phosphorylated on S592 during S-phase 525
(A-B) FANCD2 was immunoprecipitated from U2OS cells stably expressing 3xFLAG-526
FANCD2 under stringent conditions using anti-FLAG agarose. Immune complexes 527
were analyzed by immunoblotting using anti-FLAG and anti-FANCD2 antibodies (A), 528
and by staining with SimplyBlue SafeStain (B). Immunoprecipitated FANCD2 bands 529
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28
were combined and subjected to phosphoproteomic analysis using LC-MS/MS. (C) 530
HeLa cells were synchronized in M-phase by nocodazole block. Mitotic cells were 531
harvested or released into nocodazole-free medium for 12 h (S-phase) prior to 532
harvesting. (D) FANCD2 was immunoprecipated from M-phase and S-phase 533
synchronized cells using an anti-FANCD2 antibody, and visualized using silver staining 534
(D) or by immunoblotting (E). (F) A schematic of the FANCD2 protein indicating 535
phosphorylation sites identified by LC-MS/MS. (G) HeLa cells were synchronized in M-536
phase by nocodazole block. Samples were harvested and analyzed by immunoblotting 537
using an anti-FANCD2 antibody and an anti-FANCD2 pS592 phospho-specific 538
antibody. 539
540
Figure 4. Mutation of S592 disrupts FANCD2 monoubiquitination during S-phase and 541
following exposure to DNA damaging agents 542
(A) Shown is a partial model of the human FANCD2 protein, modeled on the mouse 543
Fancd2 structure (PDB #3S4W), illustrating the site of monoubiquitination K561 in red 544
and S592 in blue. (B) Site-directed mutagenesis was used to generate phospho-dead 545
(S592A) and phospho-mimetic (S592D) FANCD2 S592 variants. (C) FA-D2 cells stably 546
expressing empty vector or V5-tagged FANCD2-WT, FANCD2-S592A, or FANCD2-547
S592D were synchronized at the G1/S boundary by double-thymidine block. Cells 548
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29
were released into thymidine-free medium and whole cell lysates were analyzed by 549
immunoblotting at the indicated times post-release. 550
551
Figure S3. Mutation on S592 affects cell cycle progression / Cells lacking FANCD2 552
exhibit increased mitotic arrest in unstressed conditions 553
(A) Polyclonal populations of FA-D2 (FANCD2-/-) patient cells expressing empty vector 554
or V5-tagged FANCD2-WT, FANCD2-S592A, or FANCD2-S592D were incubated in 555
the absence (-) or presence (+) of 200 nM mitomycin C (MMC) for 24 h and whole-cell 556
lysates were analyzed by immunoblotting. (B) Wild-type HeLa and HeLa FANCD2-/- 557
cells generated by CRISPR-Cas9 gene editing were incubated in the absence or 558
presence of 100 nM MMC for 24 h. Cells were fixed and co-immunofluoresence 559
microscopy was performed for FANCD2 (green) and H3 pS10 (red). (C) The same cells 560
were treated with 100 nM MMC or 1 mM acetaldehyde for 24 h and whole-cell lysates 561
analyzed by immunoblotting. (D) Cells were incubated in the absence or presence of 562
100 nM MMC or 0.2 μM APH for 24 h. Cells were fixed, stained with propidium iodide 563
and analyzed by flow cytometry. Cell cycle stage analysis was performed using the 564
FlowJo V10.2 software. (E) FA-D2 cells stably expressing empty vector or V5-tagged 565
FANCD2-WT, FANCD2-S592A, or FANCD2-S592D were synchronized at the G1/S 566
boundary by double-thymidine block (2x dT), and then released into thymidine-free 567
medium. At the indicated time points, cells were fixed, stained with propidium iodide 568
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and analyzed by flow cytometry. Cell cycle stage analysis was performed using the 569
FlowJo V10.2 software. 570
571
Figure 5. Mutation of FANCD2 S592 leads to decreased proliferation under conditions 572
of replicative stress and increased mitotic defects 573
(A) FA-D2 cells stably expressing empty vector or V5-tagged FANCD2-WT, FANCD2-574
S592A, or FANCD2-S592D were incubated in the absence (NT) or presence of 0.4 μM 575
aphidicolin (+APH) or 20 nM mitomycin C (+MMC). Cellular proliferation was 576
monitored by measuring electrical impedance every 15 minutes over a 120 h period 577
using the xCELLigence real time cell analysis system. (B) FA-D2 cells stably expressing 578
empty vector or V5-tagged FANCD2-WT, FANCD2-K561R, FANCD2-S592A, or 579
FANCD2-S592D were incubated in the absence of any DNA damaging agents and 580
mitotic aberrations including micronuclei, nucleoplasmic bridges, and multinucleated 581
(>2) cells were scored. At least 400 cells per group were scored and the combined 582
results from two independent experiments are shown. **, P < 0.05; ***, P < 0.001. 583
584
Figure S5. Mutation of FANCD2 S592 leads to decreased proliferation under 585
conditions of replicative stress 586
(A) FA-D2 cells stably expressing empty vector or V5-tagged FANCD2-WT, FANCD2-587
S592A, or FANCD2-S592D were incubated in the absence (NT) or presence of 0.4 μM 588
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aphidicolin (+APH) or 20 nM mitomycin C (+MMC). Cellular proliferation was 589
monitored by measuring electrical impedance every 15 minutes over a 120 h period 590
using the xCELLigence real time cell analysis system. Student’s t-test was used to 591
compare electrical impedance measurements between populations at 30 h, 60 h, 90 h 592
and 120 h. (B) The same cells were incubated in the absence or presence of 2 μM 593
RO3306 and cellular proliferation was monitored by measuring electrical impedance 594
every 15 minutes over a 140 h period using the xCELLigence real time cell analysis 595
system. (C) Student’s t-test was used to compare electrical impedance measurements 596
between populations at 30 h, 60 h, 90 h, 120 h, and 140 h. 597
598
599
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745
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- + - + - + - + - + - + - + - + - +
2x Thymidine Block (dT)
λ-Phos
0 AS h post-release
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
FANCI FANCI-Ub
Cyclin A
FANCD2 FANCD2-Ub
G1/S S G2/M G1 G1/S
2 4 8 10 12 14 16
HeLa
dT Release Release
18 h
Wash
10 h
Wash
dT
18 h
Wash
- + - + - + - + - + - + α- FANCD2
1 2 3 4 5 6 7 8 9 10 11 12
α- FANCI
α- Tubulin
- - + + - - + + - - + +
FANCI FANCI-Ub
HeLa
Tubulin
FANCD2 FANCD2-Ub
λ-Phos
MMC
U2OS COS-7
α- FANCD2
1 2 3 4 5 6 7 8 9 10 11 12
α- FANCI
α- FANCA
FANCA
FANCD2 FANCD2-Ub
FANCI FANCI-Ub
- + - + - + - + - + - + λ-Phos
AS AS 0 h 2 h 0 h 2 h
FA-A (FANCA-/-) FA-A + FANCA
Tubulin α- Tubulin
AS
0 h
2 h
4 h
8 h
10 h
12 h
14 h
16 h
18 h
C
B
A
Figure 1
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Figure S1A-B
U2OS
λ-Phos
0 AS h post-release
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
FANCI FANCI-Ub
Cyclin A
FANCD2 FANCD2-Ub
G1/S S G2/M G1 G1/S
2 4 8 10 12 14 16
A
- + - + - + - + - + - + - + - + - +
2x Thymidine Block (dT)
U2OS
AS
0 h
2 h
4 h
8 h
10 h
12 h
14 h
16 h
18 h
Propidium iodide
Ce
ll n
um
be
r
B
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
M G1 G2/M S G1/S
HeLa
λ-Phos
0 AS h post-release 6 9 12 15 18
C
- + - + - + - + - + - + - + - +
Nocodazole arrest
21
FANCI FANCI-Ub
Cyclin A
FANCD2 FANCD2-Ub
Tubulin
AS
0 h
6 h
9 h
12 h
15 h
21 h
Propidium iodide
Ce
ll n
um
be
r
D
Figure S1C-D
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Figure S2A
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Figure S2B
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dT + CDKi Release Release
18 h
Wash
10 h
Wash dT + CDKi
18 h
Wash
FANCI FANCI-Ub
FANCD2 FANCD2-Ub
1 2 3 4 5 6 7 8 9 10 11 12 13 14
- + - + - + - + - + - + - +
2x Thymidine Block (dT)
λ-Phos
0 AS h post-release 2
HeLa
0 2 0 2
PURV-A SNS-032
Propidium iodide
Ce
ll n
um
be
r
2 h
0 h
AS
140
1 2 3 4 5 6 7
CDK2-Cyclin A
FANCD2
FA-D2 (FANCD2 -/-) +
1 2
FANCD2-V5 Input
IP: α-V5
FANCD2-V5
FANCD2- P
B
A
Figure 2
140
- + + + + + λ-Phos
FANCD2- P
C
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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1 2 3 4 5 6 7 8 9 10 11 12
HeLa
CDK Inhibitor
α- FANCD2
FANCD2 FANCD2-Ub
- + - + - + - + - + - + λ-Phos
NT Olo Olo II Pur Ro Rosc
1 2 3 4 5 6 7 8
α- FANCD2
α- Tubulin
HeLa
- + - + - + - + λ-Phos
NT 2 h 4 h 8 h
Purvalanol A
FANCD2 FANCD2-Ub
Tubulin
Figure S2C-D
C
D
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α-FLAG
U2OS
- + 3x FLAG
α-FANCD2 Input
α-FLAG
α-FANCD2 IP:
α-FLAG 1 2 3 4 5 6 7 8 9
200 kDa
115 kDa
3xFLAG-FANCD2 3xFLAG-FANCD2-Ub
Mw - + - B B + + + 3x FLAG-FANCD2
Input IP: α-FLAG
α-FANCD2
M
Mw 1 2 1 2 1 2 1 2 El.
1 2 3 4 5 6 7 8 9 10
AS M S
2N 4N 2N 4N 2N 4N
NOCO Release
15 h
Shake off
12 h
Harvest
1 2 3 4 5 6 7
Input
M S B M S M S
IP
FANCD2
S M S
Rabbit IgG
200 116
97 66
45
31
21
Figure 3
S8
P
S126
P
S1401 S1404 S1407 S1412 S1435 S592
P
FANCD2
P
S14XX
P
E
A B
C D
F
AS 0 12 15 18
1 2 3 4 5 6
Nocodazole
FANCD2 FANCD2-Ub
Tubulin
FANCD2-pS592
G
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S592
K561 B
A
α-H3 pS10 H3 pS10
α-Tubulin
1 2 3 4 5 6 7 8 9
α-V5 AS 0 3 6 9 12 15 18 21
Time after 2x dT release (h) FA-D2 (FANCD2-/-) + Empty
1 2 3 4 5 6 7 8 9
AS 0 3 6 9 12 15 18 21
FANCD2-V5 FANCD2-V5-Ub
Tubulin
FA-D2 + FANCD2-WT
1 2 3 4 5 6 7 8 9
α-H3 pS10
α-Tubulin
α-V5 AS 0 3 6 9 12 15 18 21
FA-D2 + FANCD2-S592A
1 2 3 4 5 6 7 8 9
Tubulin
H3 pS10
AS 0 3 6 9 12 15 18 21
FA-D2 + FANCD2-S592D
FANCD2-V5 FANCD2-V5-Ub
Figure 4
dT Release Release
18 h
Wash
10 h
Wash
dT
18 h
Wash
FANCD2
C
P
P S592D (GAT) S592A (GCA) S592 (TCA)
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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FANCD2 WT
NT
HeLa
+ MMC
FANCD2-/-
NT
+ MMC
DAPI H3 pS10 Merge
1 2 3 4 5 6
HeLa WT
FANCD2 FANCD2-Ub
H3 pS10
Tubulin
α-FANCD2
α-H3 pS10
α-Tubulin
FANCD2-/-
α- FANCI
α- CHK1 pS345 CHK1 pS345
FANCI FANCI-Ub
- - + - - + MMC - + - - + - AA 63.6
26.5 11.6
%G1 %S
%G2/M
46.9 23.6 26.0
3.97 35.5 60.1
%G1 %S
%G2/M
100 nM MMC
24.7 50.8 24.0
27.6 59.7 11.2
10.1 59.3 28.0
%G1 %S
%G2/M
NT
0.2 µM APH
HeLa WT FANCD2-/-
Figure S3A-D
B
C
D
- + - + - + - + - + - + - + - + - +
S592D
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
α-V5 MMC
FANCD2-Ub FANCD2
S592A WT Empty FA-D2
(FANCD2-/-) + A
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AS 0h 3h 6h 9h 12h 15h 18h 21h
37.7 35.5 20.2
%G1 %S
%G2/M
32.2 56.8 7.6
6.6 81.9 9.1
8.1 27.7 63.4
32.2 10.9 56.5
54.6 15.2 27.5
54.4 19.3 20.1
47.0 33.6 14.4
32.6 48.7 14.8
Time after 2x dT release (h)
FA-D2 (FANCD2-/-) + Empty
FA-D2 + FANCD2-WT
37.8 33.2 28.0
%G1 %S
%G2/M
23.0 68.3 5.8
12.2 74.0 11.6
9.3 23.2 65.8
39.2 14.7 44.8
52.9 16.4 24.6
49.2 26.9 20.2
39.1 40.7 15.3
30.9 42.2 24.3
42.6 33.1 20.1
%G1 %S
%G2/M
43.6 52.8 1.3
15.2 63.0 21.4
7.2 24.6 66.8
31.9 11.4 54.2
60.5 9.9
27.1
52.8 24.8 20.1
42.9 36.6 15.4
30.2 49.9 16.1
FA-D2 + FANCD2-S592A
34.1 35.4 23.2
%G1 %S
%G2/M
23.4 72.5 0.7
2.4 64.3 34.2
7.2 20.9 69.4
32.5 10.1 53.9
58.2 12.8 25.5
56.5 16.8 21.9
44.0 33.9 14.7
36.9 42.8 18.0
FA-D2 + FANCD2-S592D
Figure S3E
E
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0
1
2
3
Empty WT K561R S592A S592D
0.0
2.5
5.0
7.5
10.0
0 25 50 75 100 125
0.0
2.5
5.0
7.5
10.0
0 25 50 75 100 125
0.0
2.5
5.0
7.5
10.0
0 25 50 75 100 125
Ce
ll In
de
x
Empty FANCD2-WT FANCD2-S592A FANCD2-S592D
Time (h)
FA-D2 (FANCD2-/-) +
NT + APH + MMC
Figure 5
0
4
8
12
16
Empty WT K561R S592A S592D
% M
icro
nu
cle
i
Cell line
% N
ucl
eo
pla
smic
bri
dg
es
0
10
20
30
40
1 2 3 4 5%
Mu
ltin
ucl
eat
ed
ce
lls
FA-D2 (FANCD2 -/-) +
A
B ***
*** **
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NT 30 h 60 h 90 h 120 h
E-WT 0.038 0.084 0.066 0.131
E-SA 0.014 0.015 0.011 0.015
E-SD 0.913 0.292 0.061 0.053
WT-SA 0.448 0.022 0.002 0.005
WT-SD 0.064 0.363 0.811 0.296
SA-SD 0.249 0.035 0.070 0.228
0.4 µM APH
30 h 60 h 90 h 120 h
E-WT 0.359 0.377 0.088 0.015
E-SA 0.524 0.969 0.632 0.273
E-SD 0.976 0.786 0.490 0.465
WT-SA 0.116 0.176 0.029 0.025
WT-SD 0.045 0.053 0.034 0.019
SA-SD 0.389 0.625 0.724 0.635
20 nM MMC
30 h 60 h 90 h 118 h
E-WT 0.034 0.072 0.010 0.0004
E-SA 0.021 0.021 0.001 0.002
E-SD 0.009 0.173 0.003 0.006
WT-SA 0.923 0.935 0.214 0.096
WT-SD 0.085 0.177 0.280 0.052
SA-SD 0.052 0.096 0.743 0.463
Figure S5A
A
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 29, 2020. ; https://doi.org/10.1101/2020.09.29.318055doi: bioRxiv preprint
0.0
2.5
5.0
7.5
10.0
0 30 60 90 120 150
0
1
2
3
0 30 60 90 120 150
NT 30 h 60 h 90 h 120 h 140 h
E-WT 0.645 0.492 0.852 0.938 0.505
E-SA 0.265 0.059 0.027 0.135 0.152
E-SD 0.859 0.979 0.871 0.838 0.907
WT-SA 0.286 0.111 0.059 0.114 0.077
WT-SD 0.884 0.616 0.768 0.752 0.360
SA-SD 0.265 0.048 0.062 0.076 0.150
2 µM RO3306
30 h 60 h 90 h 120 h 140 h
E-WT 0.340 0.132 0.125 0.088 0.033
E-SA 0.006 0.026 0.016 0.020 0.007
E-SD 0.889 0.604 0.381 0.890 0.542
WT-SA 0.419 0.431 0.256 0.316 0.297
WT-SD 0.402 0.092 0.075 0.107 0.091
SA-SD 0.068 0.042 0.044 0.048 0.031
Empty FANCD2-WT FANCD2-S592A FANCD2-S592D
+ 2 µM RO3306
NT
Ce
ll In
de
x
Time (h)
B
C
Figure S5B-C
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 29, 2020. ; https://doi.org/10.1101/2020.09.29.318055doi: bioRxiv preprint
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 29, 2020. ; https://doi.org/10.1101/2020.09.29.318055doi: bioRxiv preprint
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 29, 2020. ; https://doi.org/10.1101/2020.09.29.318055doi: bioRxiv preprint
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 29, 2020. ; https://doi.org/10.1101/2020.09.29.318055doi: bioRxiv preprint
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 29, 2020. ; https://doi.org/10.1101/2020.09.29.318055doi: bioRxiv preprint
.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 29, 2020. ; https://doi.org/10.1101/2020.09.29.318055doi: bioRxiv preprint