Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein

Fanconi anemia (FA) is a rare autosomal and X-linked recessive disorder, characterized by congenital abnormalities, pediatric bone marrow failure and cancer susceptibility. FA is caused by biallelic mutations in any one of 16 genes. The FA proteins function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). The monoubiquitination of FANCD2 and FANCI is a central step in the activation of the FA-BRCA pathway and is required for targeting these proteins to chromatin. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. The goal of this dissertation is to study the mechanisms and regulation of FANCD2. Through this research, we have uncovered a nuclear localization signal (NLS) in FANCD2. Mutation of this NLS region impairs FANCD2 and FANCI monoubiquitination and inhibits the recruitment of FANCD2 and FANCI to chromatin. In addition, we have identified a putative CDK phosphorylation site cluster in FANCD2. Taken together, we believe these findings have enhanced our understanding of this important DNA repair protein and will allow for further investigation of this rare cancer susceptibility syndrome.


LIST OF TABLES
Laboratories using a positional gene cloning approach, following many years of collaborative efforts. [1][2][3][4] The PD20 FA patient line was originally assigned to the FA-D complementation group because of its inability to complement the mitomycin C (MMC) hypersensitivity of the FA-D reference line HSC62. [4][5][6] Using a microcellmediated chromosome transfer approach, the FANCD gene was initially mapped to 3p22-26. 4 Hejna and colleagues subsequently narrowed the critical region to ~200 kb, a region which harbored three candidate genes: TIGR-A004X28, SGC34603, and 4 AA609512. 2 Timmers and colleagues then used rapid amplification of cDNA ends (RACE) to obtain the respective full-length cDNAs and sequenced these three genes in the PD20 cells. 1 No sequence changes were uncovered in A004X28 and AA609512.
However, five sequence changes were found in SGC34603; three of which represented common polymorphisms and two represented bona fide mutations. One sequence alteration was a maternally inherited A to G transition at nucleotide 376, which resulted in a S to G missense change. This transition was also associated with missplicing and insertion of 13 nt from intron 5 into the mRNA, causing a frameshift and production of a severely truncated protein. A second paternally inherited sequence change in SGC34603 resulted in an R to H missense and hypomorphic change at position 1236 (R1236H), 1 strongly suggesting that SGC34603 was indeed the FANCD gene. Confirming this, transduction of PD20 cells with full-length wild-type FANCD rescued the MMC hypersensitivity of these cells. 1 No sequence changes in SGC34603 however were uncovered in HSC62. These findings led to the delineation of two FA-D complementation groups, FA-D1 and FA-D2, with biallelic mutations in SGC34603/FANCD2 causative for the FA-D2 complementation group. 1 Early biochemical studies of the FANCD2 protein uncovered two isoforms: an unmodified form and a higher molecular mass isoform posttranslationally modified through the covalent attachment of a single ubiquitin polypeptide, referred to as monoubiquitination. 3

FANCI
The existence of the FA-I complementation group was first established by Levitus and 5 colleagues, on the basis of somatic cell hybrid complementation analyses, 7 and the FANCI gene was subsequently identified by three groups in 2007. [8][9][10] Using a two-step genome-wide linkage approach with four genetically informative families, Dorsman and colleagues identified four candidate regions encompassing 39.4 Mb and comprising 351 genes. The sequencing of several candidate DNA repair genes failed to reveal sequence alterations. However, two strong alternative candidate genes -KIAA1794/NP_060663 and C15orf42/NP_689472 -were interrogated due to their resemblance to known FA genes in their extent of evolutionary conservation and the presence of nuclear localization signals, as many FA proteins, including FANCD2, were known to be nuclear. 3,[11][12][13][14] Sequencing of the KIAA1794 gene in all eight FA-I patients uncovered pathogenic mutations. 8 Employing a phosphoproteomics screen to identify substrates of the DNA damage response kinases ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related), the Elledge group identified KIAA1794 as an ionizing radiationinducible phosphoprotein. 15 An amino acid sequence alignment of KIAA1794/FANCI and FANCD2 uncovered a modest overall 13% identity and 20% primary sequence similarity. However, a higher degree of sequence conservation was revealed in the region encompassing KIAA1794/FANCI K523 and that flanking FANCD2 K561, the site of FANCD2 monoubiquitination. 3,10 Subsequent experiments established that, like FANCD2, FANCI is monoubiquitinated following treatment of cells with DNA damaging agents and during S phase of the cell cycle. 10 While full-length KIAA1794/FANCI protein was detected by immunoblotting in BD0952, an Epstein-Barr virus-transformed lymphocyte line from an FA patient of unassigned 6 complementation group, monoubiquitinated FANCI was not detected in these cells.
DNA sequencing revealed homozygosity for two base substitutions in KIAA1794/FANCI in BD0952 cells. These included a C to T transition leading to a P55L amino acid change, and a G to A transversion leading to a R1285Q change, the latter representing the pathogenic mutation. 10 In an alternative strategy, Sims and colleagues performed a BLAST search using the conserved amino acid sequence flanking the site of FANCD2 monoubiquitination K561, LVIRK, to identify proteins with similar monoubiquitination "consensus" sequences. 9 A highly similar sequence -LVLRKwas uncovered in the uncharacterized protein KIAA1794, which, upon further examination, displayed approximately 40% sequence similarity within the regions surrounding KIAA1794 K523 and FANCD2 K561. 9 KIAA1794/FANCI was shown to undergo DNA damage-inducible monoubiquitination, and, importantly, this monoubiquitination was dependent on both the presence and monoubiquitination of FANCD2. 9 Sims and colleagues subsequently sequenced all of the exons and intron-

FA-D2 and FA-I Clinical Aspects
FA is a clinically heterogeneous disease characterized by a wide spectrum of congenital abnormalities, a predisposition to develop early-onset progressive bone marrow failure (BMF), and an increased risk of developing solid tumors in early adulthood. The carrier frequency of FA was previously estimated at 1:300 with an expected incidence rate of approximately 1:360,000. 16 However, a more recent report cites a higher carrier frequency of 1:156 -1:209, leading to an expected incidence rate of 1:130,000. 17  clinical manifestations, and this complementation group only accounts for an estimated less than 1% of all FA cases. 18 Of the few FA-I patients with available clinical data, all have growth retardation ( Table 2). 8,9 In addition, approximately 50% of FA-I patients exhibit radial ray defects as well as both kidney and heart anomalies.  Figure 1A). The carboxyterminal EDGE motif, named for the single letter amino acid code, is not required for FANCD2 monoubiquitination or nuclear foci formation. However, this motif is necessary for complementation of the ICL-sensitivity of FA-D2 patient cells. 25 An interaction between FANCD2 and the major DNA processivity factor PCNA has also been described. 26 13 14 FANCD2 and FANCI in chromatin, and for efficient ICL repair. 27 In addition, the amino terminal 58 amino acids of FANCD2 harbors a nuclear localization signal (NLS). 28 This sequence is required for the efficient nuclear localization of both FANCD2 and FANCI, and for their monoubiquitination and function in ICL repair. 28 FANCI is a 1,328 amino acid protein with a molecular mass of 149 kDa ( Figure 1B). FANCI has an armadillo (ARM) repeat domain, which form superhelical folds involved in mediating protein-protein interactions. 10 Figure 1C). Multiple positively charged grooves within these troughs are capable of accommodating single and double-stranded DNA. The sites of monoubiquitination are located in the ID2 interface within solvent accessible tunnels.
However, these tunnels are predicted to be too small to accommodate the ubiquitin ligase machinery, raising challenging questions about the temporal, kinetic, and spatial aspects of FANCD2 and FANCI monoubiquitination. 35

FANCD2 and FANCI Monoubiquitination
Protein ubiquitination is a multi-step post-translational modification that results in the covalent attachment of a single ubiquitin molecule (monoubiquitination) or chain of linked ubiquitin molecules (polyubiquitination) to a lysine residue on a substrate protein. Mono-and poly-ubiquitination signal a wide range of cellular instructions, the former being most typically associated with the targeting of proteins to specific subcellular localizations, while the canonical role of the latter is to target proteins for degradation by the proteasome. 36,37 In the general process of ubiquitination, an E1 The monoubiquitination of FANCD2 and FANCI is required for their assembly into discrete nuclear foci, where they co-localize with several proteins including the protein product of one of the major breast and ovarian cancer susceptibility genes BRCA1. 3,9,10 (Figure 2). 59 SCF, along with the anaphase-promoting complex or cyclosome (APC/C), plays an intricate and critical role in cell-cycle regulation. 60 The F-box protein of the SCF complex functions in substrate recognition and recruitment. 59 Figure 3A). 27 Thus, FANCD2 and FANCI might be shielded from further ubiquitination via intermolecular association. It is also possible that monoubiquitination promotes an intramolecular association between ubiquitin covalently attached to K561 and the amino-terminal CUE domain, 27 potentially blocking the ubiquitin on K561 from further ubiquitination, similar to that for EPS15 ( Figure 3B). The latter model would more closely align with recent findings from the Sobeck laboratory indicating that activation of the FA-BRCA pathway coincides with dissociation of FANCD2 and FANCI. 74 ID2 dissociation is triggered by ATM/ATRmediated phosphorylation of a cluster of at least six FANCI SQ/TQ motifs, and is followed by the monoubiquitination of FANCD2, see below. 33,74 The NEDD4 E3 ubiquitin ligase can mono-and polyubiquitinate substrates, and the balance between these two posttranslational modifications is determined, at

FANCD2 and FANCI Deubiquitination
The site-specific monoubiquitination of FANCD2 and FANCI is a reversible process. The USP1 gene is also transcriptionally repressed upon cellular exposure to DNA damaging agents, a process that requires the function of the p21 cyclin-dependent kinase inhibitor. 87,91 Both negative regulatory mechanisms facilitate the timely accumulation of monoubiquitinated FANCD2 and FANCI following the induction of DNA damage. 89

FANCD2 Phosphorylation
The FANCD2 protein is also subject to posttranslational modification by phosphorylation. The ATM kinase is a member of the phosphatidylinositol-3-OHkinase-like family of protein kinases (PIKK). Bilallelic mutations in the ATM gene cause the autosomal recessive disorder Ataxia-telangiectasia (AT). 92 AT is clinically characterized by progressive cerebellar ataxia, telangiectasias, immune defects, and increased susceptibility to hematologic cancers of B and T cell origin, as well as 29 central nervous system tumors. 93,94 The ATM kinase plays a major coordinating role in the cellular response to DNA DSBs and phosphorylates multiple substrates to halt cell cycle progression and initiate DNA repair. For example, the ATM kinase phosphorylates T68 of the CHK2 kinase following exposure to ionizing radiation (IR) to initiate cell cycle arrest at the G1-S boundary. 95 In 2002, Taniguchi and colleagues discovered that ATM phosphorylates FANCD2 on S222, S1401, S1404, and S1418 in response to IR exposure. 96 Phosphorylation of FANCD2 S222 is required for the establishment of the IR-inducible S phase checkpoint, an ability to pause DNA synthesis in the presence of chromosomal DSBs. 97 However, ATM-mediated FANCD2 S222 phosphorylation is dispensable for FANCD2 monoubiquitination, nuclear foci formation, and ICL resistance. 96 An important role for the ATR kinase in the regulation of FANCD2 has also been established. 98,99 While ATM primarily responds to DNA DSBs, ATR is a major regulator of the DNA replication checkpoint, primarily responding to agents that disrupt DNA replication, e.g. hydroxyurea, an inhibitor of deoxyribonucleotide reductase, aphidicolin (APH), a processive DNA polymerase inhibitor, and UV irradiation, which promotes the formation of cyclobutane pyrimidine dimers. 100,101 Mutations in ATR underlie Seckel syndrome (SCKL1) a rare autosomal recessive disorder characterized by severe growth retardation, short stature, microcephaly, mental retardation, and increased risk for acute myeloid leukemia (AML), myelodysplasia (MDS), and aplastic anemia, similar to FA. [102][103][104] Following exposure to DNA crosslinking agents, ATR and FANCD2 co-localize in nuclear foci. 98 Phosphorylation could also promote the interaction of FANCD2 with DNA in chromatin, which has been shown to lead to increased efficiency of monoubiquitination. 56, 106

FANCI Phosphorylation
FANCI was originally identified as an ATM/ATR substrate in a large-scale SILAC (stable isotope labeling with amino acids in cell culture) proteomics screen. 15 As described above, six S/TQ motifs are positioned proximal to K523, the sites of monoubiquitination. These S/TQ sites become phosphorylated in the absence of prior monoubiquitination, indicating that this posttranslational modification acts upstream of monoubiquitination. 33 Importantly, mutation of these S/TQ sites results in abrogation of FANCI and FANCD2 monoubiquitination and nuclear foci formation, 33 31 indicating that phosphorylation of the FANCI S/TQ cluster functions as a molecular switch to activate the FA-BRCA pathway. However, the underlying molecular mechanism remains unclear. Recently, it has been suggested that activation of the pathway coincides with disassociation of the ID2 heterodimer, and that ID2 disassociation is triggered by ATM/ATR-mediated phosphorylation of the S/TQ cluster. 106 Using cell-free Xenopus laevis egg extracts, Sareen and colleagues demonstrated that a FANCI S/TQ cluster phosphorylation-dead mutant failed to dissociate from FANCD2 and failed to undergo monoubiquitination. Conversely, a phosphorylation-mimetic mutant failed to associate with FANCD2 and exhibited increased monoubiquitination. 106 Taken together, these findings indicate that phosphorylation of FANCI at the conserved S/TQ cluster is a key mechanism in the activation of the FA-BRCA pathway. The introduction of a cluster of negatively charged phosphate groups may result in a localized charge repulsion that promotes ID2 disassociation, enabling access of the previously occluded ubiquitin ligase machinery.

Replisome Surveillance
Early studies indicated that, in addition to being monoubiquitinated following exposure to DNA damaging agents, FANCD2 and FANCI monoubiquitination also occurs during unperturbed cell cycle progression. In addition, monoubiquitinated FANCD2 co-localizes with BRCA1 and RAD51 in nuclear foci during S phase. 97 These findings strongly suggested that monoubiquitinated FANCD2 might play an important role in maintaining genome stability during the process of DNA replication. 10,97 Accordingly, the FANCD2 and FANCA proteins are required for the maintenance of common chromosomal fragile site (CFS) stability. 107 CFS are chromosomal loci that are prone to breakage when cells are cultured under conditions of DNA replication stress, for example following treatment with low concentrations of the DNA polymerase inhibitor APH. 108 CFSs are hot spots for sister chromatid exchanges (SCE), chromosomal translocations, and viral integration, and are frequently rearranged or deleted in cancer. 109 The physical localization of FANCD2 and FANCI to FRA3B and FRA16D, the most frequently expressed CFSs, has also been demonstrated. 110 Combined immunofluorescence microscopy and FISH analysis of metaphase chromosomes revealed the presence of paired FANCD2/I-stained CFSs on sister chromatids linked by BLM helicase-associated ultra-fine DNA bridges (UFBs). 110,111 These paired FANCD2/I sister foci form during S phase and persist into mitosis, and their presence strongly suggest that FANCD2 and FANCI participate in the resolution of stalled or collapsed DNA replication forks that recurrently arise at particularly unstable genomic loci. 107,110,111 In further support of an important role for FANCD2 in the resolution of stalled or collapsed replication forks, Fancd2-/-MEFs are ICL hypersensitive yet do not display increased sensitivity to IR. 23 Consistent with a role for the FA-BRCA pathway in the DNA replication stress response, FANCD2 has been shown to interact with several components of the DNA replisome. For example, as mentioned earlier, FANCD2 interacts with the DNA polymerase processivity factor PCNA, via a conserved PIP-box, and this interaction is necessary for efficient FANCD2 monoubiquitination and ICL repair. 26 In addition, using a method called iPOND (isolation of proteins on nascent DNA), the Cortez group recently discovered that FANCD2 and FANCI, in addition to ATR, MRE11 and other proteins, are highly enriched at stalled and collapsed replication forks following depletion of deoxyribonucleotide pools. 112 Interestingly, FANCI, and not FANCD2, was also shown to accumulate at active replication forks prior to fork staling, suggesting common and independent functions for these proteins. 112

Independent Functions of FANCD2 and FANCI BLM Complex Chromatin Assembly
Since the discovery of FANCI, it has generally been accepted that FANCD2 and FANCI function cooperatively in the resolution of ICLs. However, several recent studies have suggested independent functions for these proteins. For example, it has recently been determined that FANCD2, and not FANCI, is an important regulator of the recruitment of the BLM helicase to chromatin. 130 Biallelic mutations in the BLM gene underlie Bloom syndrome (BS), a rare recessive disorder characterized by growth retardation, sunlight sensitivity, telangiectasia, hypo-or hyper-pigmentation of the skin, and increased susceptibility to hematologic cancers. 131 The BLM helicase is a member of the RecQ family of helicases, which also includes the WRN and RECQ4 proteins, and possesses ATP-dependent 3"-5" DNA helicase activity. 132

Nucleosome Assembly Activity
FANCD2 was recently identified in a proteomics screen of histone H3/H4-interacting proteins. 135 Recombinant FANCD2 was subsequently confirmed to bind to H3/H4 in vitro. 135 These findings suggested that FANCD2 might play an active role in chromatin dynamics and/or reorganization. Indeed, using a topological assay and a nucleosome assembly assay, FANCD2 was shown to promote nucleosome assembly to an extent comparable to that of the known nucleosome assembly protein Nap1.
Similar to the promotion of BLM complex chromatin localization, FANCD2 histone binding and nucleosome assembly activity appear to be FANCI and monoubiquitination-independent. 135  Upon DSB generation, TIP60 is rapidly recruited to sites of damage where it acetylates multiple substrates including H2A, H4, p53, and ATM. [139][140][141][142] Menin has also been shown to function in chromatin remodeling, and DNA repair. 136,143,144 Cells lacking menin are more sensitive to DNA damage and the interaction between FANCD2 and menin is increased following exposure to IR. 136 Collectively, these findings suggest that FANCD2 may function in a chromatin remodeling capacity during the early stages of ICL repair. Similarly, the Zhang group purified full-length FANCI and demonstrated that it can bind to multiple DNA substrates, including HJs, dsDNA, ssDNA, and flap structures, without any apparent structural preference. 31 Longerich et al also demonstrated that purified FANCI is capable of binding to dsDNA, ssDNA, and HJs. 80 Importantly, however, co-purified ID2 displayed a much lower affinity for dsDNA and ssDNA, yet maintained a robust binding activity towards HJs and various branched DNA structures. 31 Consistent with these findings, the Pavletich group described the structure of co-crystals of FANCI bound to splayed Y DNA at a resolution of 7.8 Å. 35 Based on this structure and the structural homology between FANCD2 and FANCI, the authors proposed that the ID2 heterodimer could have two sets of pseudo-symmetrical dsDNA/ssDNA binding sites, structures that could arise upon stalling of one or two replication forks at an ICL. 35 Notably, the Patel group discovered that, in addition to binding to dsDNA and ssDNA, FANCD2 exhibits 3" to 5" exonucleolytic activity towards ssDNA in the presence of Mg 2+ or Mn 2+ . 164

Conclusions
The well as FAAP100, FAAP24, and FAAP20 [4,5]. Following monoubiquitination, FANCD2 and FANCI co-localize in discrete nuclear foci with several well characterized DNA repair proteins including BRCA1 and NBS1 [6][7][8][9]. However, importantly, the domain structure, regulation, and function of both FANCD2 and FANCI are poorly described. Furthermore, the mechanism(s) by which FANCD2 and FANCI are localized to the nucleus remains unknown.
Several studies have established that the FANCD2 and FANCI proteins function proximal to or within chromatin [6,[8][9][10]. As FANCD2 and FANCI are relatively large proteins, with molecular weights of 164 and 149 kDa, respectively, the nuclear import of these proteins necessitates an energy-dependent active transport mechanism. The most common nuclear protein transport mechanism involves the 70 recognition of nuclear localization signals (NLSs) by members of the importin (Imp) superfamily of nuclear transporters, followed by translocation through the nuclear pore complexes [11]. NLSs generally comprise short stretches of basic amino acids either alone (monopartite) or separated by one (bipartite) or two (tripartite) mutation-tolerant linker regions of 10-12 amino acids (bipartite). NLSs in the cargo protein are recognized by the Imp α subunit of the Imp α/β heterodimer or by Imp β alone, and nuclear transport is coupled to GTP hydrolysis [11].
In this study, we describe the identification and functional characterization of a NLS in the amino terminus of FANCD2. We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of green fluorescent protein (GFP). In addition, using deletion and site-directed mutagenesis strategies we establish that the amino terminal 58 amino acids of FANCD2 are necessary for the nuclear and chromatin localization of FANCD2 in FA-D2 patient-derived cells.
Importantly, we also demonstrate that the nuclear and chromatin localization of a subset of the cellular pool of FANCI is dependent on the nuclear import of FANCD2.

FANCD2 contains a highly conserved amino-terminal nuclear localization signal, which facilitates nuclear expression of GFP
In silico analysis using cNLS mapper uncovered several high-scoring Imp α/dependent bipartite NLSs within the amino-terminal 58 amino acids of FANCD2 (Figs. S1A and B) [12]. In contrast, cNLS mapper did not predict any high scoring NLSs in FANCI. A sequence alignment of FANCD2 from multiple species illustrates strong evolutionary conservation in general (Fig. S1C) uniform cytoplasmic and nuclear fluorescence (Fig. 1B). Conversely, cells transiently expressing D2-1-58-GFP primarily exhibited nuclear fluorescence (Fig. 1B). Similar findings were observed with IMR90 cells (Fig. S1D). These results demonstrate that the amino terminal 58 amino acids of FANCD2 are necessary to promote exclusive nuclear GFP localization. In support of an importin /-dependent mechanism of 74 nuclear import, treatment with ivermectin, a broad-spectrum inhibitor of importin /dependent nuclear import [13], inhibited the exclusive nuclear localization of D2-1-58-GFP, herein referred to as D2-NLS-GFP (Figs. S1E and F). In addition, mass spectrometry analysis of FANCD2 immune complexes revealed the presence of importin 1, as well as the nuclear pore complex proteins NUP160 and NUP155 (Supplemental Table 1). Using a chromatin fractionation approach we also observed that the majority of GFP-WT resided in a soluble cytoplasmic and nuclear fraction (S) (Fig. S1G, lane 5). While a large proportion of D2-NLS-GFP also resided in a soluble cytoplasmic and nuclear fraction, a higher relative proportion of D2-NLS-GFP was detected in a chromatin-associated nuclear fraction (C) (Figs. S1G, lane 9 and H).
Taken together these results demonstrate that the amino-terminal 58 amino acids of FANCD2 harbors a bona fide NLS that can promote exclusive nuclear GFP localization.

The FANCD2 NLS is required for the nuclear localization of FANCD2
To determine the functional significance of the FANCD2 NLS, we next generated deletion and missense mutations of this amino acid sequence. Two amino-terminal deletion mutations, FANCD2-N57, lacking amino acids 2-58, and FANCD2-N100, lacking amino acids 2-101, were generated ( Fig. 2A). In addition, using a site-directed mutagenesis approach, amino acids K4, R5, and R6, the most highly conserved basic amino acids within this region (Fig. 1A), were mutated to N4, N5, and N6, herein referred to as FANCD2-3N (Fig. 2A). These FANCD2 cDNAs were cloned into the pLenti6.2 lentiviral vector, which contains a carboxy-terminal V5 tag, and lentivirus  2B). In contrast, nuclear and focal localization of wild type FANCD2 was largely resistant to permeabilization (Fig. 2B). Similar findings were obtained with FA-D2 cells expressing FANCD2-N100 (Fig. 2C). In addition, a partial yet significant defect in nuclear localization was observed for the FANCD2-3N mutant, compared to wild type FANCD2 (Fig. 2C).

The FANCD2 NLS is required for the nuclear localization of a subset of FANCI
Next, we examined the sub-cellular localization of FANCI in our FA-D2 cell series. It is important to note that the FA-D2 cells used in this study, like all known FA-D2 patient-derived lines, harbor hypomorphic FANCD2 mutations and express residual FANCD2 protein [15]. IF analysis revealed that FANCI was uniformly present in both the cytoplasm and nucleus of FA-D2 cells in the absence or presence of MMC (Figs.   3A and B). Importantly, complementation of FA-D2 cells with wild type FANCD2  3A and B). Furthermore, MMC-inducible FANCI nuclear foci formation was restored in these cells, consistent with previous studies [8,9] (Fig. 3A). Similar results were obtained with hTERT-immortalized mutant and FANCD2-complemented KEAE FA-D2 patient-derived cells [15] (Fig. S2A). In contrast to wild type FANCD2, both the FANCD2-N57 and -N100 NLS mutants failed to promote the exclusive nuclear localization of FANCI (Figs. 3B and S2B). Furthermore, a partial defect in FANCI nuclear localization was observed for FA-D2 cells expressing FANCD2-3N (Fig. 3B).
To rule out the possibility that deletion of the amino-terminus of FANCD2 prevented its heterodimerization with FANCI, we tested the ability of the FANCD2-N57 mutant to physically interact with FANCI by transiently transfecting COS-7 cells with GFP-tagged FANCI and V5-tagged FANCD2-WT or FANCD2-N57 and examining their ability to interact by co-immunoprecipitation. Co-expression of both FANCD2-WT and FANCD2-N57 with FANCI led to its stabilization (Fig. 3C, upper panel,   lanes 3 and 4). Furthermore, the FANCD2-N57 mutant was capable of interacting with FANCI and, when corrected for levels of protein input, no appreciable difference in the level of interaction between FANCI and FANCD2-N57 and FANCI and FANCD2-WT was observed (Fig. 3C, lower panels, lanes 3 and 4). Together, these results establish that the nuclear localization of a subset of FANCI is dependent in part on FANCD2, and suggest a piggyback mechanism of nuclear FANCI targeting that is dependent on the FANCD2 amino-terminal NLS.

The FANCD2 NLS is required for efficient FANCD2 and FANCI monoubiquitination and chromatin association
Next, we examined the consequences of disruption of the FANCD2 NLS on the monoubiquitination and chromatin localization of FANCD2 and FANCI. Similar to the FANCD2-K561R mutant that cannot be monoubiquitinated [6], mutation of the FANCD2 NLS had a significant impact on its monoubiquitination: the N57 and N100 NLS deletion mutants failed to undergo spontaneous or MMC-inducible FANCD2 monoubiquitination (Fig. 4A, lanes 7-10). A faint band of similar molecular weight to wild type FANCD2 can be seen in the -FANCD2 panel for FA-D2 cells expressing FANCD2-N57 and -N100 (Fig. 4A, lanes 7-10). This is most likely the hypomorphic FANCD2-R1236H missense mutant (see Materials and Methods) [14], as this band is also present in FA-D2 cells expressing LacZ (lanes 1 and 2) and is not recognized by the anti-V5 antibody (Fig. 4, lanes 7-10). The FANCD2-3N mutant also exhibited considerably reduced spontaneous and MMC-inducible monoubiquitination (Fig. 4A, lanes 11 and 12). Previous studies have demonstrated that FANCD2 and FANCI monoubiquitination are interdependent [8,9]. Accordingly, FANCI monoubiquitination was largely abrogated in FA-D2 cells stably expressing the N57 and N100 NLS deletion mutants, similar to that observed in cells expressing FANCD2-K561R (Fig. 4A, lanes 5-10). A very modest statistically insignificant decrease in FANCI monoubiquitination was also observed for FA-D2 cells expressing the FANCD2-3N mutant (Fig. 4A, lanes 11 and 12). In addition, a chromatin fractionation approach revealed that the FANCD2-N57, -N100, and -3N mutants were largely defective in chromatin localization compared to FANCD2-WT nM for FANCD2-N57, -N100, and -K561R compared with wild type FANCD2) (Fig. S4). Recent studies have demonstrated increased error-prone nonhomologous DNA end joining (NHEJ) in FA patient cells [17]. Therefore, we also examined the recruitment of DNA-PK CS to nuclear foci in FA-D2 cells expressing LacZ, wild type

87
FANCD2 or the FANCD2 N57 mutant, using an antibody raised against DNA-PK CS phosphorylated on S2056, a marker of NHEJ [17,18]. Persistent increased DNA-PK CS pS2056 nuclear foci formation was observed in FA-D2 cells expressing LacZ following treatment with MMC, and this phenotype was rescued by wild type FANCD2 (Fig. 5B). In contrast, we observed markedly increased MMC-inducible DNA-PK CS pS2056 nuclear foci formation in FA-D2 cells expressing FANCD2 N57, compared to cells expressing LacZ or wild type FANCD2 (Fig. 5B). Taken together, these results demonstrate that the FANCD2 NLS is essential for the correct function of the FA-BRCA pathway in the cellular ICL response.

Discussion
Despite their critical role in the cellular ICL response and their tumor suppressor function, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins. For FANCD2, a large 1451 amino acid protein, only two functional motifs, a PCNA-interaction motif, or PIP box, and a carboxy-terminus EDGE motif, have been described to date [10,19]. Our laboratory has also recently identified and characterized a CUE ubiquitin-binding domain in the amino-terminus of FANCD2, which mediates noncovalent binding to ubiquitin, and is essential for efficient cellular ICL repair [20]. In this study we describe the functional characterization of an amino terminal FANCD2 NLS. While a previous study reported the existence of a FANCD2 NLS, this study failed to examine the functional consequences of its disruption in a FA-D2 patient-derived cell system [21].  [22,23], unconventional bipartite NLSs with extended linker lengths have also been described [24][25][26]. However, cNLS mapper searches for both conventional and unconventional bipartite NLSs and only detected the former [12]. In addition to monopartite and bipartite NLSs, at least two other classes of NLS have been described: tripartite containing three clusters of basic amino acids similar to those found in L-periaxin and the epidermal growth factor receptor (EGFR) family [27,28], as well as NLSs containing dispersed basic residues within a random coil structure such as that found for 5-lipoxygenase [29]. These NLSs are poorly characterized in comparison with their mono-and bi-partite counterparts and are not predicted by cNLS mapper or PSORT II amino acid prediction algorithms. While the crystal structure of the murine Fanci-Fancd2 heterodimer (ID2) has been solved, the majority of the NLS described in this study was not crystallized precluding speculation about the structure of this region [30]. It is also important to note that FANCD2 harbors several putative phosphorylation sites within the amino terminal 58 amino acids (PhosphoSitePlus), which may also contribute to the regulation of its nuclear localization [31].
Our studies suggest that FANCD2 is imported to the nucleus via an importin /-dependent mechanism as treatment with ivermectin, a broad-spectrum inhibitor of importin /-dependent nuclear import [13], results in markedly decreased exclusive nuclear localization of D2-NLS-GFP. Furthermore, using mass spectrometry we have recently detected importin 1, as well as the nuclear pore complex proteins NUP160 and NUP155, in FANCD2 immune complexes (Supplemental Table 1 [32]. While loss of this NLS reduced FANCI nuclear accumulation, this NLS was not completely necessary for FANCI or FANCD2 nuclear accumulation, strongly suggesting the existence of alternative nuclear import mechanisms for both proteins, consistent with our data [32]. The elucidation of the crystal structure of the ID2 heterodimer indicates that the FANCD2 and FANCI NLSs are spatially separated within this structure [30], arguing against the simultaneous contribution of both NLSs to nuclear import of the ID2 complex. Taken together, these results suggest that FANCI localizes to the nucleus via FANCD2-independent and -dependent mechanisms (Fig. 6). These findings are also consistent with the observation that only a minor fraction of the cellular pools of FANCD2 and FANCI physically interact [8,9], reinforcing the concept of ID2 complex-independent functions for both proteins, such as that recently described by Chaudhury and colleagues [33]. A recent study has also established that a fraction of FANCD2 is transported to the nucleus following MMC exposure via an indirect interaction with importin 4 (IPO4), which is mediated by the C/EBP transcription factor [34]. While clearly important for ICL repair, this mechanism in unlikely to be the major mechanism of FANCD2 nuclear import as 91 robust levels of nuclear FANCD2 were observed in C/EBP-null mouse embryonic fibroblasts as well as cells depleted of IPO4 and C/EBP [34]. Nevertheless, this C/EBP/IPO4-dependent FANCD2 nuclear import mechanism could account for the low levels of nuclear FANCD2-N57 and FANCD2-N57 observed in our studies.
Interestingly, we observed markedly increased MMC-inducible chromosome aberrations and DNA-PK CS pS2056 nuclear foci formation in FA-D2 cells expressing  . 4A, top panel) and is predicted to retain residual or partial function. Indeed, the vast majority of FA-D2 patient-derived cells retain residual FANCD2 function with complete loss of FANCD2 predicted to result in embryonic lethality [15]. Our results suggest that the FANCD2-N57 mutant interferes with residual FANCD2 R1236H function, perhaps competing with FANCD2 R1236H for heterodimerization with FANCI, or in a manner analogous to missense p53 mutations, by assembling into nonfunctional homo-oligomers, the formation of which has been suggested by previous studies [30,35].
Based on our findings, and those of several other groups, we propose the following model for early steps in the FA-BRCA pathway of ICL repair (Fig. 6). A subset of the total cellular pools of FANCD2 and FANCI associate in the cytoplasm to assemble into the ID2 heterodimer. The ID2 heterodimer is transported to the nucleus most likely via an importin /-mediated transport process, using the amino terminal (red) in the cytoplasm, and that the ID2 heterodimer is transported to the nucleus via an importin α/β (brown)-mediated transport mechanism, using the amino terminal FANCD2 NLS (light green). Nuclear ID2 binds to DNA (orange) and is also phosphorylated by the ATM/ATR kinases (dark green). One or both of these events may trigger ID2 complex restructuring, facilitating FANCD2 and FANCI monoubiquitination by FANCL (black), UBE2T (yellow) and the FA core complex (not shown).
94 NLS of FANCD2. Once inside the nucleus the ID2 heterodimer is targeted to sites of ICL damage possibly via the association of FANCD2 with PCNA and the replication fork machinery [19]. Recent in vitro studies have demonstrated that FANCI binding to DNA is necessary for robust stimulation of the monoubiquitination of FANCD2 [36].
However, analysis of the ID2 crystal structure indicates that the FANCD2 K561 side chain, the site of monoubiquitination, is embedded within the ID2 interface [30].
Furthermore, a solvent accessible tunnel adjacent to FANCD2 K561 is predicted to be too small to accommodate the active site of the UBE2T ubiquitin-conjugating enzyme [30,37]. Therefore, either 1) monoubiquitination occurs on FANCD2 and FANCI monomers prior to ID2 heterodimerization or 2) binding of the ID2 complex to DNA leads to a conformational change in the ID2 structure leading to the exposure of K561R and FANCI K523, and their subsequent monoubiquitination, as has been proposed [36]. A recent study by Sareen and colleagues suggests that activation of the FA-BRCA pathway coincides with dissociation of FANCD2 and FANCI [38]. ID2 dissociation is triggered by ATR/ATM-mediated phosphorylation of a cluster of at least six FANCI SQ motifs, and is followed by the monoubiquitination of FANCD2 [38,39]. Once monoubiquitinated, FANCD2 can then facilitate the recruitment of several structure specific nucleases, including FAN1 and FANCP/SLX4, initiating the process of ICL removal [40][41][42][43][44][45][46]. Invitrogen).

Plasmids, site-directed mutagenesis, and transient transfections
The full length, N57, and N100 FANCD2 cDNA sequences were TOPO cloned into the pENTR⁄D-TOPO (Invitrogen) entry vector, and subsequently recombined into the pLenti6.2/V5-DEST (Invitrogen) destination vector and used to generate lentivirus for the generation of stable cell lines. The FANCD2-KRR4NNN (FANCD2-3N)  FANCI proteins [4,5]. This central activation step promotes FANCD2 and FANCI colocalization to discrete nuclear foci with several downstream DNA repair proteins, including FANCS/BRCA1 and NBS1 [6][7][8][9]. Monoubiquitinated FANCD2 has been shown to recruit the structure specific endonucleases FAN1 and FANCP/SLX4, which suggests that this modified form of FANCD2 functions to promote one or more incision steps during the ICL repair process [10,11]. FANCD2 and FANCI are also posttranslationally modified by phosphorylation in response to DNA damage by ATM (Ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related). ATM phosphorylates FANCD2 in response to IR exposure independently of monoubiquitination [12]. Phosphorylation of FANCD2 by ATR promotes FANCD2 monoubiquitination in response to exposure to ICL-inducing agents and is important in its function in ICL repair [13]. FANCI phosphorylation by ATM/ATR on six SQ/TQ residues has been linked to activation of the FA-BRCA pathway, potentially by promoting the dissociation of the FANCD2-FANCI heterodimer [14,15].
Although the main focus concerning FANCD2 has been based on DNA damage activation, FANCD2 has also been shown to be monoubiquitinated during unperturbed cell cycle progression in S phase, where it co-localizes with BRCA1 and RAD51 [16]. With the addition of hydroxyurea (HU), which arrests DNA replication, FANCD2 has been shown to protect stalled replication forks from degradation by MRE11, and to promote replication restart at stalled replication forks by recruiting and interacting with CtIP (CtBP-interacting protein) [17,18]. These studies suggest that FANCD2 has many roles outside of the more recognized DNA damage inducible function, specifically during the cell cycle.
In this study, we have determined that FANCD2 is phosphorylated in a DNA damage-independent manner, which we believe is essential for the regulation of FANCD2 function during unperturbed S phase. Many DNA repair proteins are regulated by cyclin-dependent kinase (CDK) mediated phosphorylation, such as BRCA1 and NBS [19,20]. Here we show that FANCD2 is a substrate of CDK through immunoprecipitation studies as well as CDK inhibitor experiments. Importantly, we have discovered and are beginning to characterize a putative CDK site cluster in FANCD2 that is positioned proximal to FANCD2s site of monoubiquitination.
Missense mutations of these sites yields unexpected results, a phospho-mimetic mutant fails to induce a response to DNA replication or damaging agents and a phospho-dead mutant is constitutively activated. The findings from this study establish novel phosphorylation of FANCD2 in unperturbed S phase and reveal a CDK phosphorylation site cluster that is posttranslationally modified to negatively regulate FANCD2 monoubiquitination.

FANCD2 is phosphorylated in the absence of DNA damage
Initial findings from a previous study using FANCD2 patient cells expressing FANCD2-WT, FANCD2-K561R, and truncation mutants, suggested that FANCD2 may be phosphorylated in the absence of DNA damage (data not shown). In order to analyze the phosphorylation of FANCD2 under these conditions, a lambda phosphatase assay was performed. Lambda phosphatase cleaves phosphorylated serine, threonine, and tyrosine residues [21]. HeLa, U2OS, COS7, and BJ-TERT cells were treated with and without MMC and lysates were incubated in the presence and absence of lambda phosphatase (Fig. 1A). When comparing incubation with and without lambda phosphatase, the electrophoretic mobility of FANCD2 increases in both the presence and absence of MMC, indicating that FANCD2 is phosphorylated independent of DNA damage. It is interesting to note that this is not seen with FANCI.
To ensure this finding is due to phosphorylation, a lambda phosphatase assay was done using HeLa, FA-D2 patient-derived cells stably express the empty vector, FANCD2-WT, and FANCD2-K561R, which were treated for the indicated times with lambda phosphatase (Fig. 1B). Reaction products were then run on a Phos-tag gel; Phos-tag is a novel phosphate-binding tag that binds phosphorylated proteins [22].
Treatment with lambda phosphatase causes the stabilization of non-phosphorylated FANCD2 and limits the isoforms present when compared to lack of treatment. To ensure the molecular weight difference in FANCD2 was due to the cleavage of phosphate groups and not an unforeseen interaction with the lambda phosphatase enzyme, protein phosphatase 1 (PP1) enzyme was used (Fig. 1C). This enzyme indicates that FANCD2 is phosphorylated on several residues, in a DNA damageindependent manner.

FANCD2 phosphorylation is maximal in S phase
It is known that FANCD2 is activated through monoubiquitination during S phase of the cell cycle; however, the regulation behind this cycle dependent posttranslational modification is currently unknown [16]. To determine if FANCD2 is differentially phosphorylated throughout the cell cycle, several cell synchronizations were performed. Using HeLa cells, a nocodazole block was performed, blocking the cells in early M phase ( Fig. 2A). Cells were then released for the indicated times and cell pellets were separated into three, treating with and without lambda phosphatase for 2 h, and the third was fixed and stained with propidium iodide for FACS analysis.
Although there is a molecular weight shift difference when looking at FANCD2 via western blot, this shift in migration is most prominent at 12 and 15 h post-release, correlating with S phase of the cell cycle. Again, when probing for FANCI, no molecular weight shift was observed. Cyclin A was used as a cell cycle marker, as it is active and upregulated in S and G2 phases of the cell cycle [23]. To explore FANCD2 phosphorylation during the cell cycle, a second synchronization method was performed. Using both HeLa (Fig. 2B) and U2OS cells (Fig. S1), a double thymidine block arrest was used to synchronize in G1/S phase of the cell cycle. A lambda 117 phosphatase assay and FACs analysis was then performed, and in a similar manner as the nocodazole block, a maximal molecular weight shift difference is seen 0-4 h postrelease. This is indicative of S phase of the cell cycle, suggesting there is a DNA damage independent phosphorylation of FANCD2 that correlates with its monoubiquitination.

Non-DNA damage inducible phosphorylation of FANCD2 is independent of ATM, ATR, and the core complex member FANCA
It is known that FANCD2 and FANCI are phosphorylated by the DNA damage response kinases ATM and ATR [12][13][14]. To test if ATM was the kinase responsible for the phosphorylation of FANCD2 in S phase seen via the lambda phosphatase assay, PEBS (ATM-/-) and YZ5 (ATM+/+) fibroblasts were used for a double thymidine block and subsequent lambda phosphatase assay, as described previously (Fig. 3A). If ATM is essential for this posttranslational modification, the molecular weight difference in FANCD2 treated with and without lambda phosphatase would be absent or reduced. In comparing PEBS and YZ5 cells, there is no difference in FANCD2s electrophoretic mobility with the addition of the lambda phosphatase enzyme, indicating this phosphorylation is largely independent of ATM. To determine ATRs importance in FANCD2s phosphorylation in the absence of DNA damage and in S phase of the cell cycle, 066 Seckel patient lymphoblasts (ATR-/-) and wild-type lymphoblasts were then used (Fig. 3B). Similar to the results seen in ATM deficient cells, ATR does not appear to be crucial for this mobility in FANCD2. The FA core complex is responsible for orchestrating FANCD2s monoubiquitination;  . S2). FANCD2 K561R is unable to become monoubiquitinated; however, a molecular weight shift difference is still seen. This indicates that monoubiquitination is not essential for FANCD2s phosphorylation.

FANCD2 is a potential substrate of CDK phosphorylation
Cell cycle progression is regulated through phosphorylation by CDKs [24]. We next assessed whether FANCD2 was a substrate of CDK phosphorylation by using FA-D2 patient cells stably expressing LacZ-V5 or FANCD2-V5 and performing an immunoprecipitation (IP) (Fig. 4A). V5 agarose was used to pull down FANCD2-V5.
A pan phospho serine antibody was then used, which recognizes phosphorylated substrates of CDK, and FANCD2 is seen in the FA-D2 patient cell line expressing FANCD2. A second IP was performed using a different cell system, U2OS cells stably expressing 3XFLAG tagged FANCD2 (Fig. 4B). Here we used FLAG agarose and performed an IP with U2OS and U2OS 3X FLAG-FANCD2 fibroblasts. Similar results were obtained as with FA-D2 FANCD2-V5; FANCD2 is a substrate of CDK as seen through the pan phospho serine antibody. The U2OS 3XFLAG FANCD2 IP was then run on a commassie gel in triplicate and the FANCD2 bands were combined and a tryptic digestion was performed in preparation for mass spectrometry (Fig. S3).
Using TiO 2 phosphopeptide enrichment, these samples were analyzed for phospho peptides (Fig. 4C). Several known ATM/ATR sites were found in this sample, as well as one serine protein site, S692, which resides in the phosphoCDK cluster proximal to

Treatment with CDK inhibitor purvalanol A alters FANCD2s phosphorylation shift
As it was determined that FANCD2 is a potential substrate of CDK, we wanted to establish which CDK was responsible. If maximal FANCD2 phosphorylation occurs during S phase of the cell cycle, CDK2 could be the potential kinase responsible. In order to determine a specific CDK was responsible for FANCD2s non-DNA damaging phosphorylation, various CDK inhibitors with differing specificities were utilized (Fig. 5A). HeLa cells were incubated with each indicated inhibitor for 24 h and a lambda phosphatase assay was performed to determine if a specific CDK is required for FANCD2s phosphorylation (Fig. 5B). Two chemicals that comprise CDK2

FANCD2 contains a conserved putative CDK consensus site cluster
As the previous results suggest that FANCD2 is a substrate of CDK, the FANCD2 sequence was analyzed for potential CDK phosphorylation sites. A multiple sequence alignment of FANCD2 found strong evolutionary conservation in a cluster of three S/P sites: S525, S624, S726 (Fig. 6A). Interestingly, when looking at the murine crystallized structure of FANCD2, this putative CDK cluster is situated in close proximity to FANCD2s monoubiquitination site (Fig. 6B). As previous results have established a connection between maximal FANCD2 phosphorylation occurring during S phase, which is when FANCD2 is monoubiquitinated during the cell cycle, the position could shed light on FANCD2s activation. It is important to note that the S/P site found through mass spec analysis, S692, is also situated within this cluster, albeit on a short uncrystallized loop. To determine the functional significance of the putative CDK cluster, we generated two missense mutants in S525, S624, S726. These sites were mutated into either phospho-dead triple alanine (TA) or phospho-mimetic triple aspartic acid (TD) mutants. The FANCD2 cDNAs were cloned into the pLenti6.2 lentiviral vector that contains a carboxy-terminal V5 tag. FA-D2 patientderived cells were used to stably express the empty vector, wild type FANCD2 and the missense mutants. To determine the consequences of mutating the putative CDK site cluster, the FA-D2 cell lines were then treated with and without MMC to determine their monoubiquitination efficiency (Fig. 6C). Although these mutations did not abrogate FANCD2 monoubiquitination, the TA mutant is constitutively activated and the TD mutant is unable to elicit a monoubiquitination response with the addition of damage. FANCI monoubiquitination is unaffected by the mutation of the FANCD2 phosphorylation sites. To further study the functional effect of mutating these conserved phosphorylation residues, FA-D2 + empty, FANCD2-WT, TA, and TD cells were incubated with and without either HU or aphidicolin (APH) (Fig. S5). As we believe these residues are important in non-DNA damage inducible activation of FANCD2, it is important to explore DNA replication inhibitors, as FANCD2 is active during S phase of the cell cycle. HU transiently stalls replication by causing an imbalance in the deoxyribonucleoside triphosphate pool and APH inhibits DNA polymerase α [25,26]. Similar to the results obtained when treating with MMC, the TA mutant appears to be constitutively monoubiquitinated, while the TD mutant fails to become monoubiquitinated following exposure to the replicative inhibitors.
Interestingly, when looking at CHK1 pS345 and RPA pS3/4, both mutants have an increase without the addition of treatment. These are markers for blocked DNA replication, suggesting that mutating these phosphorylation sites causes inefficient DNA replication. Taken together, these results demonstrate the importance of these sites in FANCD2 function and further analysis must be performed to determine their mechanism of action and specific purpose in DNA replication.

Discussion
Although FANCD2 has been shown to function in unperturbed S phase of the cell cycle, how this is regulated has not been studied [16]. In this study, with the use of lambda phosphatase assays, we have revealed novel DNA damage-independent phosphorylation of FANCD2. Several cell lines show FANCD2 having an increase in electrophoretic mobility through an SDS Page gel with the addition of lambda phosphatase, indicating the cleavage of phosphate groups from the protein during incubation. This variance is not seen in FANCI, even after the addition of MMC.
Although we know FANCI is phosphorylated by ATM/ATR with the addition of damage, this inability to visualize a molecular weight shift difference, due to phosphate cleavage, could be explained by how extensive the phosphorylation is [15].
Currently, six S/TQ sites have been described to be phosphorylated by ATM/ATR [14]. The limited number of residues phosphorylated could be the reason for no visible shift difference seen in this assay. FANCD2 and FANCI are very large proteins, 1451 and 1328 amino acids, respectively [27]. In order to see this slight shift in protein molecular weight, due to their size, the immunoblot must be extensively run and the ability of FANCD2 phosphorylation to be visualized could mean it is extensively phosphorylated. It is important to note that the shift is also seen with incubation with the second phosphatase, PP1. Interestingly, we have found that maximal FANCD2 phosphorylation, as seen through the lambda phosphatase assay, occurs at S phase.
This has been visualized by double thymidine block, as well as a nocodazole block.
This suggests that FANCD2s function in the cell cycle could be regulated through phosphorylation.
DNA damage inducible FANCD2 phosphorylation has been widely studied, so it is necessary to determine if the key regulatory proteins involved in this well-known posttranslational modification are essential for the non-DNA damaging regulation of FANCD2 [12,16,28]. With the use of patient fibroblast and lymphoblasts, we found that the extensive phosphorylation of FANCD2, in S phase, is unaffected by the absence of ATM, ATR, and FANCA. Due to FANCD2s monoubiquitination in S phase and, now seen, phosphorylation, we next wanted to determine if these posttranslational modifications were reliant on one another [16]. In using FA-D2  [19,20]. In using two different cell systems and an antibody, that specifically recognizes substrates of CDKs through its known consensus sequence; we found that FANCD2 is potentially a substrate of CDK. To try to determine which residues are phosphorylated and narrow down our sites of interest, we used the U2OS 3XFLAG FANCD2 immunoprecipitation to enrich for phospho peptides and analyze by mass spectrometry. Several ATM/ATR sites were enriched, however, a putative CDK consensus site that has not been studied due to its lack of sequence conservation was also found, S592. We then optimized several CDK inhibitors to try to focus on a specific CDK responsible. Correlating with our finding that FANCD2s phosphorylation is maximal during S phase, the inhibitor Purvalanol A seemed to limit FANCD2s molecular weight shift with the addition of lambda phosphatase. This inhibitor is specific towards CDK2, with some inhibition of CDK1 [29]. We further compared the CDK1 specific inhibitor, RO-3306, and Purvalanol A and found that incubation with the CDK2 inhibitor for 4 and 8 h limited FANCD2s phosphorylation.
This suggests that FANCD2s phosphorylation during S phase is regulated by CDK2.
Through initial sequence and structural analysis, several important putative CDK phosphorylation sites have been discovered. The CDK S/P residues S525, S624, and S726 were highly conserved amongst various species and are situated proximal to FANCD2s monoubiquitination site and form a cluster with the mass spectrometry phospho peptide found, S592. This site, however, is located on a short uncrystallized loop in FANCD2. Through site-directed mutagenesis, S525, S624, S726 were mutated to alanines (TA), phospho-dead, or aspartic acid (TD), phospho-mimetic, residues.

Antibodies and immunoblotting
For immunoblotting analysis, cell pellets were washed in PBS and lysed in 2% w/v SDS, 50 mM Tris-HCl, 10 mM EDTA followed by sonication for 10 s at 10% immunoblotting or stained using Colloidal Blue Staining Kit (Invitrogen) for mass spectrometry.

Plasmids, site-directed mutagenesis, and transient transfections
The An EDGE motif was found to be essential for ICL-sensitivity [1]. A PIP-motif was discovered to be necessary for the interaction between FANCD2 and PCNA and is required for efficient FANCD2 monoubiquitination and repair [2]. A CUE ubiquitin binding domain in FANCD2 was needed for interaction with FANCI, as well as, efficient ICL repair [3]. It was known that FANCD2 functions in the nucleus; however, no experiments prior to my graduate research had examined how FANCD2 is transported for ICL repair.
One project that was comprehensively studied during my graduate research, focused on the identification and characterization of a putative NLS in the amino terminus of FANCD2 [4]. In this research, through initially fusing a predicted NLS region to GFP, we discovered that the first 58 amino acids of FANCD2 are required to promote GFP nuclear localization. This region contained two bipartite NLSs, however, both were found to be essential for localization. As mentioned, FANCD2 and FANCI are phosphorylated by ATM/ATR in response to DNA damaging agents in order to facilitate the repair of DSBs [5][6][7].
However, it has been demonstrated that FANCD2 is monoubiquitinated during unperturbed S phase of the cell cycle and functions to protect stalled replication forks from MRE11 degradation [8,9]. FANCD2s DNA damage-independent regulation has not been described. Through the utilization of lambda phosphatase assays in my most recent project, I found that FANCD2 is extensively phosphorylated, specifically in S 146 phase of the cell cycle. This led us to hypothesize that this is how FANCD2 is being regulated in the absence of damage. In synchronizing various cell lines, we found that this phosphorylation pattern is independent of the major DNA damage kinases, ATM and ATR, as well as the core complex member FANCA. Interestingly, we also showed that the ability of FANCD2 to become monoubiquitinated did not alter FANCD2s phosphorylation and the monoubiquitinated form of FANCD2 persists even with the addition of the phosphatase enzyme. This demonstrates that the DNA damage independent phosphorylation of FANCD2 is also independent of its monoubiquitination. As this phosphorylation is maximal at S phase, we found that FANCD2 is indeed a substrate of CDK phosphorylation. To narrow down which residues in FANCD2 could be phosphorylated by CDK, we found several conserved SP sites that were surprisingly clustered around FANCD2s site of monoubiquitination.
These sites were then mutated to phospho-dead alanine (TA) residues or phosphomimetic asparagine (TD) residues. Currently, we have found that these sites do alter FANCD2s ability to become monoubiquitinated with the addition of MMC or DNA replication inhibitors. The TA mutant is constitutively monoubiquitinated and the TD mutant does not elicit a response. These preliminary findings suggest that these sites are phosphorylated in order to inhibit FANCD2s monoubiquitination. These FANCD2 mutants need to be further studied to determine the function of FANCD2 monoubiquitination inhibition. Ultimately, due to the importance of FANCD2 in aspects of DNA repair and DNA replication, this protein should be further characterized to potentially discover significant therapeutic implications for FA that allow for tailored prophylactic and chemotherapeutic measures to be adopted.

APPENDIX II
Supplemental information for manuscript II: "Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal"

Generation of FANCD2-GFP fusion vectors
To fuse amino acids 1-27 of FANCD2 to the amino terminus of GFP to generate D2-1-27-GFP, we PCR amplified the coding sequence of amino acids 1-27 using the following forward and reverse primers: FP, 5"-AAAGAGCTCCACCATGGTTTCC-