CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic … · 2020. 9. 29. · 19 Fanconi anemia, FANCD2, CDK phosphorylation, cell cycle, ubiquitination 20 21 Abstract 22 Fanconi

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CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity 1

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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

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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

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*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

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.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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

https://doi.org/10.1101/2020.09.29.318055

http://creativecommons.org/licenses/by/4.0/

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Keywords 18

Fanconi anemia, FANCD2, CDK phosphorylation, cell cycle, ubiquitination 19

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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

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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

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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

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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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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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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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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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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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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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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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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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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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27

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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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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PMID: 26633632. 744

745



https://doi.org/10.1101/2020.09.29.318055


- + - + - + - + - + - + - + - + - +

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



https://doi.org/10.1101/2020.09.29.318055


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

- + - + - + - + - + - + - + - + - +


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



https://doi.org/10.1101/2020.09.29.318055


Figure S2B



https://doi.org/10.1101/2020.09.29.318055


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

- + - + - + - + - + - + - +


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



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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)



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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



https://doi.org/10.1101/2020.09.29.318055


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



https://doi.org/10.1101/2020.09.29.318055


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



https://doi.org/10.1101/2020.09.29.318055


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



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