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• W1969127943 abstract "•A comprehensive resource of proteins recruited to stalled replication forks•FANCD2 binds the MCM2–MCM7 replicative helicase upon ATR signaling•FANCD2 controls replisome function under stressful conditions•Human primary cells depend on FANCD2 to ensure normal cell proliferation Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2–MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis. Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2–MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis. Oncogenic proliferation occurs without sufficient nucleotides to support DNA replication, resulting in replication failure at common fragile sites and the formation of DNA double-strand breaks (Aird et al., 2013Aird K.M. Zhang G. Li H. Tu Z. Bitler B.G. Garipov A. Wu H. Wei Z. Wagner S.N. Herlyn M. Zhang R. Suppression of nucleotide metabolism underlies the establishment and maintenance of oncogene-induced senescence.Cell Rep. 2013; 3: 1252-1265Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, Bester et al., 2011Bester A.C. Roniger M. Oren Y.S. Im M.M. Sarni D. Chaoat M. Bensimon A. Zamir G. Shewach D.S. Kerem B. Nucleotide deficiency promotes genomic instability in early stages of cancer development.Cell. 2011; 145: 435-446Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, Jones et al., 2013Jones R.M. Mortusewicz O. Afzal I. Lorvellec M. García P. Helleday T. Petermann E. Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication stress.Oncogene. 2013; 32: 3744-3753Crossref PubMed Scopus (174) Google Scholar). The formation of replication-associated DNA lesions distinguishes cancer cells from normal cells (Bartkova et al., 2005Bartkova J. Horejsí Z. Koed K. Krämer A. Tort F. Zieger K. Guldberg P. Sehested M. Nesland J.M. Lukas C. et al.DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.Nature. 2005; 434: 864-870Crossref PubMed Scopus (2234) Google Scholar, Gorgoulis et al., 2005Gorgoulis V.G. Vassiliou L.V. Karakaidos P. Zacharatos P. Kotsinas A. Liloglou T. Venere M. Ditullio Jr., R.A. Kastrinakis N.G. Levy B. et al.Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1693) Google Scholar), and failure to correctly sense or rescue stalled replication intermediates is the underlying cause of several chromosomal instability syndromes such as Fanconi anemia (FA), an inherited disorder characterized by congenital abnormalities, bone marrow failure, and cancer proneness (Alter et al., 2003Alter B.P. Greene M.H. Velazquez I. Rosenberg P.S. Cancer in Fanconi anemia.Blood. 2003; 101: 2072Crossref PubMed Scopus (124) Google Scholar, Tischkowitz and Hodgson, 2003Tischkowitz M.D. Hodgson S.V. Fanconi anaemia.J. Med. Genet. 2003; 40: 1-10Crossref PubMed Scopus (205) Google Scholar). Biallelic inactivation of any of 16 FA genes confers cellular hypersensitivity to chemotherapeutic interstrand DNA crosslinking agents (Deans and West, 2011Deans A.J. West S.C. DNA interstrand crosslink repair and cancer.Nat. Rev. Cancer. 2011; 11: 467-480Crossref PubMed Scopus (703) Google Scholar). The products of FA genes orchestrate the coordinated action of translesion DNA polymerases, endonucleases, and homologous recombination activities for repairing DNA crosslinks (Kim and D’Andrea, 2012Kim H. D’Andrea A.D. Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway.Genes Dev. 2012; 26: 1393-1408Crossref PubMed Scopus (387) Google Scholar). During the activation of the FA pathway, FANCD2 and FANCI are coupled to an ubiquitin moiety by the FA core complex (Garcia-Higuera et al., 2001Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D’Andrea A.D. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway.Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar, Smogorzewska et al., 2007Smogorzewska A. Matsuoka S. Vinciguerra P. McDonald 3rd, E.R. Hurov K.E. Luo J. Ballif B.A. Gygi S.P. Hofmann K. D’Andrea A.D. Elledge S.J. Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair.Cell. 2007; 129: 289-301Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), which includes eight proteins (FANCA–FANCG, FANCL, and FAAP100). This posttranslational modification is catalyzed by the E3 ligase FANCL and is necessary for the DNA repair function of FANCD2 (Meetei et al., 2003Meetei A.R. de Winter J.P. Medhurst A.L. Wallisch M. Waisfisz Q. van de Vrugt H.J. Oostra A.B. Yan Z. Ling C. Bishop C.E. et al.A novel ubiquitin ligase is deficient in Fanconi anemia.Nat. Genet. 2003; 35: 165-170Crossref PubMed Scopus (471) Google Scholar). Whereas transformed FA cell lines appear uniquely hypersensitive to interstrand DNA crosslinking agents, there is strong evidence that FANC proteins are mobilized in order to maintain genome stability in response to a variety of replication obstacles (Chan et al., 2009Chan K.L. Palmai-Pallag T. Ying S. Hickson I.D. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis.Nat. Cell Biol. 2009; 11: 753-760Crossref PubMed Scopus (437) Google Scholar, Howlett et al., 2005Howlett N.G. Taniguchi T. Durkin S.G. D’Andrea A.D. Glover T.W. The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability.Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (225) Google Scholar, Langevin et al., 2011Langevin F. Crossan G.P. Rosado I.V. Arends M.J. Patel K.J. Fancd2 counteracts the toxic effects of naturally produced aldehydes in mice.Nature. 2011; 475: 53-58Crossref PubMed Scopus (377) Google Scholar, Naim and Rosselli, 2009Naim V. Rosselli F. The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities.Nat. Cell Biol. 2009; 11: 761-768Crossref PubMed Scopus (243) Google Scholar, Rosado et al., 2011Rosado I.V. Langevin F. Crossan G.P. Takata M. Patel K.J. Formaldehyde catabolism is essential in cells deficient for the Fanconi anemia DNA-repair pathway.Nat. Struct. Mol. Biol. 2011; 18: 1432-1434Crossref PubMed Scopus (140) Google Scholar, Schlacher et al., 2012Schlacher K. Wu H. Jasin M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.Cancer Cell. 2012; 22: 106-116Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). Here, we used unbiased proteomic screens to identify proteins that bind nascent DNA as well as proteins that associate with FANCD2 in response to nucleotide depletion. We present evidence that FANCD2 is a general effector of ATR signaling that targets stalled replication forks and associates with MCM2–MCM7 hexamers upon DNA replication stress. In human primary dermal fibroblasts (HDFs), FANCD2 is necessary for limiting replication-associated defects and recovering from replication inhibition by nucleotide depletion. We propose that FANCD2 participates in a general surveillance mechanism that controls the replication machinery under stressful conditions. We used isolation of protein on nascent DNA (iPOND) to identify proteins bound to nascent DNA in the vicinity of the replisome (Kliszczak et al., 2011Kliszczak A.E. Rainey M.D. Harhen B. Boisvert F.M. Santocanale C. DNA mediated chromatin pull-down for the study of chromatin replication.Sci. Rep. 2011; 1: 95Crossref PubMed Scopus (45) Google Scholar, Lopez-Contreras et al., 2013Lopez-Contreras A.J. Ruppen I. Nieto-Soler M. Murga M. Rodriguez-Acebes S. Remeseiro S. Rodrigo-Perez S. Rojas A.M. Mendez J. Muñoz J. Fernandez-Capetillo O. A proteomic characterization of factors enriched at nascent DNA molecules.Cell Rep. 2013; 3: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, Sirbu et al., 2011Sirbu B.M. Couch F.B. Feigerle J.T. Bhaskara S. Hiebert S.W. Cortez D. Analysis of protein dynamics at active, stalled, and collapsed replication forks.Genes Dev. 2011; 25: 1320-1327Crossref PubMed Scopus (299) Google Scholar). Newly synthesized DNA was labeled for 5 min with EdU, coupled to biotin azide, and pulled down along with DNA-bound proteins for mass spectrometry (MS) analyses. For each protein identified, we calculated the ratio of normalized spectral counts detected after EdU pulse labeling versus thymidine chase (Piersma et al., 2010Piersma S.R. Fiedler U. Span S. Lingnau A. Pham T.V. Hoffmann S. Kubbutat M.H. Jiménez C.R. Workflow comparison for label-free, quantitative secretome proteomics for cancer biomarker discovery: method evaluation, differential analysis, and verification in serum.J. Proteome Res. 2010; 9: 1913-1922Crossref PubMed Scopus (117) Google Scholar) and plotted pulse:chase ratios in increasing order (Figure S1 available online). Among the top 100 proteins enriched 3- to 30-fold on nascent DNA near replication forks, we identified RFC, PCNA, and DNA polymerases and minichromosome maintenance (MCM) proteins as well as a number of replication-associated factors (Figure S1). The entire data set of three iPOND-MS replicates is available in Table S1. Overall, more than 70% of proteins on nascent DNA identified in a similar study were rediscovered here (Lopez-Contreras et al., 2013Lopez-Contreras A.J. Ruppen I. Nieto-Soler M. Murga M. Rodriguez-Acebes S. Remeseiro S. Rodrigo-Perez S. Rojas A.M. Mendez J. Muñoz J. Fernandez-Capetillo O. A proteomic characterization of factors enriched at nascent DNA molecules.Cell Rep. 2013; 3: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Next, we exploited the robustness of the iPOND-MS methodology to identify proteins that bind nascent DNA specifically upon nucleotide depletion. Cells treated with 1 mM hydroxyurea (HU) were labeled for 50 min with EdU in order to yield EdU-substituted replication tracts with a mean length comparable to that of 5 min labeling in untreated cells (Luke-Glaser et al., 2010Luke-Glaser S. Luke B. Grossi S. Constantinou A. FANCM regulates DNA chain elongation and is stabilized by S-phase checkpoint signalling.EMBO J. 2010; 29: 795-805Crossref PubMed Scopus (72) Google Scholar). We calculated the average ratio of normalized spectral counts obtained by iPOND-MS from three replicates of HU-treated versus -untreated cells (Table S1). The ratio (±HU) of spectral counts for RFC4 and the catalytic subunit of polymerase δ was now close to 1, whereas PCNA was underrepresented in samples from HU-treated cells (Figures 1A and 1C ). This confirmed that, in comparison to untreated cells (5 min EdU), the amount of replication factors isolated from HU-treated cells (50 min EdU) was not increased. By contrast, the FA proteins FANCI and FANCD2 were identified among the most highly enriched proteins at stalled replication forks along with the DNA damage checkpoint proteins ATR and MRE11 and the sister chromatin cohesion proteins SMC1A, SMC3, and STAG2. In total, we detected 30 proteins enriched at stalled forks with high confidence in three independent replicates (Figure 1B and Table S1). A STRING analysis for known and predicted protein-protein interactions (http://string.embl.de) revealed that a significant proportion of proteins that target stalled forks form an interconnected network (Figure 1B). To confirm the specific association of FANCD2 and FANCI with nascent DNA in response to fork stalling, we coupled iPOND with western blotting. Whereas the isolation of PCNA with EdU-substituted DNA was reduced in HU-treated cells, FANCD2 and FANCI near or at stalled replication forks were detected on nascent DNA exclusively upon treatment with HU (Figure 1C), in agreement with the MS data analysis (Table S1). These results indicate that FANCD2 and FANCI associate closely with newly synthesized DNA. To gain insights into the mechanism of FANCD2 recruitment to nascent DNA, we sought to identify FANCD2-interacting proteins in cells treated with HU. We created a stable human embryonic kidney (HEK) 293T cell line expressing FANCD2 fused to a FLAG epitope at its amino terminus. HEK 293T FLAGFANCD2 cells were exposed to HU for 1 hr, and chromatin was prepared in native conditions. Chromatin-bound proteins were solubilized by treatment with the pan-nuclease benzonase, fractionated by size exclusion, and purified by affinity on anti-FLAG agarose (Figures 2A and 2B ). The complete list of FLAGFANCD2 chromatin-associated proteins identified by MS is shown in Table S2; it includes several proteins identified by iPOND in the surroundings of the replisome, such as FANCI, a confirmed partner of FANCD2 (Smogorzewska et al., 2007Smogorzewska A. Matsuoka S. Vinciguerra P. McDonald 3rd, E.R. Hurov K.E. Luo J. Ballif B.A. Gygi S.P. Hofmann K. D’Andrea A.D. Elledge S.J. Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair.Cell. 2007; 129: 289-301Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), as well as MSH2 and MSH6 (Figure 2C), consistent with a previous report (Williams et al., 2011Williams S.A. Wilson J.B. Clark A.P. Mitson-Salazar A. Tomashevski A. Ananth S. Glazer P.M. Semmes O.J. Bale A.E. Jones N.J. Kupfer G.M. Functional and physical interaction between the mismatch repair and FA-BRCA pathways.Hum. Mol. Genet. 2011; 20: 4395-4410Crossref PubMed Scopus (42) Google Scholar). In addition, we identified a number of peptides derived from all of the subunits of the MCM2–MCM7 replicative helicase as well as multiple subunits of replication factor C (Figure 2C). We also identified the FACT complex (SUPT16H and SSRP1) among FANCD2-associated factors. FACT interacts with MCM2–MCM7 hexamers to facilitate DNA replication initiation and elongation (Abe et al., 2011Abe T. Sugimura K. Hosono Y. Takami Y. Akita M. Yoshimura A. Tada S. Nakayama T. Murofushi H. Okumura K. et al.The histone chaperone facilitates chromatin transcription (FACT) protein maintains normal replication fork rates.J. Biol. Chem. 2011; 286: 30504-30512Crossref PubMed Scopus (60) Google Scholar, Tan et al., 2006Tan B.C. Chien C.T. Hirose S. Lee S.C. Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication.EMBO J. 2006; 25: 3975-3985Crossref PubMed Scopus (132) Google Scholar) and has been identified on nascent DNA near replication forks (Table S1; Lopez-Contreras et al., 2013Lopez-Contreras A.J. Ruppen I. Nieto-Soler M. Murga M. Rodriguez-Acebes S. Remeseiro S. Rodrigo-Perez S. Rojas A.M. Mendez J. Muñoz J. Fernandez-Capetillo O. A proteomic characterization of factors enriched at nascent DNA molecules.Cell Rep. 2013; 3: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). These data indicate that, upon translocation to the chromatin compartment, FANCD2 associates with components of the replication machinery. First, to examine the association of FANCD2 with MCM2–MCM7 in more detail, we confirmed the presence of MCM proteins in FLAGFANCD2 preparations by western blotting (Figure 2D). FLAG peptides interfered by competition with the pull-down of FLAGFANCD2 and the associated MCM proteins, thereby confirming that the proteins were isolated specifically with the FLAG antibody (Figure 2D). To guarantee that the formation of the FANCD2-MCM complex was not a consequence of protein aggregation, which may result from FLAGFANCD2 overexpression, we probed the association of endogenous MCM and FANCD2 proteins in HEK 293T cells treated for 1 hr with HU. Chromatin extracts were prepared and fractionated by gel filtration on Superdex 200 (Figure 2E, left). Next, fractions containing FANCD2 and MCM proteins were pooled and supplemented with EtBr and DNase I in order to disrupt protein interactions mediated by bridging DNA molecules. In these conditions, FANCD2 was successfully immunoprecipitated with an antibody against MCM3 along with MCM2, MCM5, and MCM7 (Figure 2E, right). The MCM5 antibody presumably recognizes MCM5 exclusively within MCM3–MCM5 subcomplexes, given that it pulled down only the MCM3 and MCM5 proteins. Reciprocally, MCM proteins were pulled down with a FANCD2 antibody (Figure 2E, right). Finally, we fractionated chromatin extracts from untreated and HU-treated HEK 293T cells by gel filtration on superose 6 in order to improve the resolution of high-molecular-weight species. FANCD2 from untreated cells did not coelute with MCM proteins (Figure 2F, top). However, after treatment with HU, the amount of FANCD2 in the chromatin fraction increased as expected, and its fractionation profile changed dramatically (Figure 2F, bottom). FANCD2 both eluted with a large macromolecular complex in the exclusion volume of the column, and coeluted with MCM proteins as part of a smaller protein complex. This suggests that a fraction of FANCD2 associates with MCM proteins in response to nucleotide depletion. Incidentally, the hydrodynamic properties of MCM protein complexes did not change after treatment with HU. One possibility is that MCM proteins associate with a distinct set of proteins before and after HU, forming different protein complexes with similar Stokes radii. Next, we monitored the dynamics of FANCD2-MCM2–MCM7 complex formation. Chromatin extracts were prepared during the course of treatment with HU for 4 hr. As expected, the amount of FLAGFANCD2 immunoprecipitated from chromatin increased 30 min after exposure to HU and remained constant thereafter (Figure 3A). By contrast, the induced association of FANCD2 and MCM proteins was transient and maximal after 60 min in HU (Figure 3A). Checkpoint kinases control replisome function through the phosphorylation of multiple replication proteins (De Piccoli et al., 2012De Piccoli G. Katou Y. Itoh T. Nakato R. Shirahige K. Labib K. Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases.Mol. Cell. 2012; 45: 696-704Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, Ilves et al., 2012Ilves I. Tamberg N. Botchan M.R. Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex.Proc. Natl. Acad. Sci. USA. 2012; 109: 13163-13170Crossref PubMed Scopus (29) Google Scholar). Here, we used Ser108 pMCM2, a posttranslational modification catalyzed by ATR (Cortez et al., 2004Cortez D. Glick G. Elledge S.J. Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases.Proc. Natl. Acad. Sci. USA. 2004; 101: 10078-10083Crossref PubMed Scopus (262) Google Scholar), to mark replisomes that have been targeted by the S phase checkpoint. We detected Ser108 pMCM2 in FLAGFANCD2 pull-downs (Figure 3A), and a significant proportion of human HDFs exposed to HU exhibited subnuclear foci containing both FANCD2 and Ser108 pMCM2 (Figure 3B), indicating that FANCD2 associates with MCM proteins upon S phase checkpoint signaling. FANCD2 and Ser108 pMCM2 also partially colocalized in HDFs exposed to the DNA polymerases inhibitor aphidicolin or to the interstrand DNA crosslinking agent mitomycin C (Figure S2A). FANCD2 and Ser108 pMCM2 colocalized constitutively in the osteosarcoma cell line U2OS and upon the transformation of human dermal fibroblasts with the E6 and E7 oncoproteins from human papillomavirus 16 (Figure S2A), which induced constitutive activation of ATR signaling (Figure S2B). These observations suggest that the transient association of FANCD2 with MCM proteins is a general response to replication stress signaling induced by oncogenic transformation or by replication-blocking agents. To test whether ATR-Chk1 signaling was necessary for FANCD2 to target MCM proteins, we used ETP-46464, a specific inhibitor of ATR (Toledo et al., 2011Toledo L.I. Murga M. Zur R. Soria R. Rodriguez A. Martinez S. Oyarzabal J. Pastor J. Bischoff J.R. Fernandez-Capetillo O. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations.Nat. Struct. Mol. Biol. 2011; 18: 721-727Crossref PubMed Scopus (374) Google Scholar). ATR inhibition partially reduced the phosphorylation of MCM2 on Ser108 and completely abolished the phosphorylation of Chk1 on Ser345 (Figure 3C, upper panel). ATR inhibition also impaired the association of FLAGFANCD2 with MCM proteins in response to treatment with HU (Figure 3C, lower panel). By contrast, the formation of the FANCD2-MCM2–MCM7 complex was preserved upon Chk1 inhibition with SB218078 (Figure 3D), whereas phosphorylation of the Chk1 target CDC25C on Ser216 was completely abolished (Sanchez et al., 1997Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25.Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1123) Google Scholar). Thus, ATR activity, but not Chk1 activity, is necessary for promoting a transient association between FANCD2 and MCM proteins. ATR is required for FANCD2 monoubiquitination and the retention of Ub-FANCD2 in chromatin (Andreassen et al., 2004Andreassen P.R. D’Andrea A.D. Taniguchi T. ATR couples FANCD2 monoubiquitination to the DNA-damage response.Genes Dev. 2004; 18: 1958-1963Crossref PubMed Scopus (336) Google Scholar). To test whether the monoubiquitination of FANCD2 was necessary for its association with MCM proteins, we generated an HEK 293T cell line that expressed FLAGK561R-FANCD2, a nonmonoubiquitinable FANCD2 mutant protein. Whereas FLAGK561R-FANCD2 bound chromatin very weakly and transiently during HU treatment, it did interact with Ser108 pMCM2 and MCM5 (Figure 3E). FANCD2 also pulled down Ser108 pMCM2 and MCM5 from FA complementation group A (FA-A) cells exposed to HU (Figure 3F) and colocalized with Ser108 pMCM2 foci in FA-A cells (Figure S2C). As expected, the fraction of chromatin-bound FANCD2 increased dramatically in FA-A cells (PD220) complemented with a complementary DNA (cDNA) encoding FANCA, thereby improving the yield of FANCD2 and MCM immunoprecipitations (Figure 3F). It is noteworthy that FANCD2-MCM-containing complexes were also detected in untreated FA-A cells complemented with FANCA (Figure 3F), most likely as a consequence of constitutive replication stress induced by the transformation of the FA-A cell line with SV40. Collectively, these results suggest that, in response to ATR signaling, FANCD2 binds transiently to MCM proteins independently of its monoubiquitination by the FA core complex. Next, we used the baculovirus heterologous expression system to test whether FANCD2 can interact directly with either one of the MCM proteins. FLAGFANCD2 pulled down wild-type MCM4, MCM5, and MCM7 proteins, whereas the FLAGFANCD2 and MCM3 coimmunoprecipitation was inconclusive, and MCM2 and MCM6 were undetectable in FLAGFANCD2 immunoprecipitates (Figure 3G). This result indicates that FANCD2 targets the replisome via direct binding to a subset of MCM proteins. Recent evidence indicated that the S phase checkpoint directly inhibits replisome function rather than maintaining replisome stability (De Piccoli et al., 2012De Piccoli G. Katou Y. Itoh T. Nakato R. Shirahige K. Labib K. Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases.Mol. Cell. 2012; 45: 696-704Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, Sabatinos et al., 2012Sabatinos S.A. Green M.D. Forsburg S.L. Continued DNA synthesis in replication checkpoint mutants leads to fork collapse.Mol. Cell. Biol. 2012; 32: 4986-4997Crossref PubMed Scopus (45) Google Scholar). To test whether FANCD2 is necessary for controlling the replisome, we visualized replication tracts by DNA fiber labeling in HDFs transfected with small interfering RNAs (siRNAs). HDFs were synchronized by serum starvation and released into S phase, and replication tracks were labeled with consecutive pulses of 20 min with the thymidine analogs IdU and CldU. To monitor DNA chain elongation from isolated replication forks, we measured the length of CldU replication tracts adjacent to IdU tracts. The knockdown of FANCD2 had no significant impact on the median length of CldU tracts in unchallenged HDFs (Figure S3), consistent with a previous study (Song et al., 2010Song I.Y. Barkley L.R. Day T.A. Weiss R.S. Vaziri C. A novel role for Fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells.Cell Cycle. 2010; 9: 2375-2388Crossref PubMed Scopus (17) Google Scholar). By contrast, CldU tracks labeled for 2 hr in the presence of HU were significantly longer in FANCD2-depleted cells than in cells treated with control siRNA (Figures 4A and 4B and Table S3). Thus, FANCD2 is necessary for actively restraining DNA synthesis upon nucleotide depletion. Within replicons, sister forks from one origin progress coordinately at a similar rate (Conti et al., 2007Conti C. Saccà B. Herrick J. Lalou C. Pommier Y. Bensimon A. Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells.Mol. Biol. Cell. 2007; 18: 3059-3067Crossref PubMed Scopus (160) Google Scholar). To gain further evidence of uncontrolled replisome movement in FANCD2-depleted cells, we analyzed the progression of sister forks labeled in red during HU treatment that emanate from a replication origin labeled in green before treatment with HU (Figure 4C). In control cells, more than 65% of sister red tracks had a length ratio close to 1, consistent with symmetrical fork movement. In contrast, the proportion of sister red tracks with a similar length drop to 30% in FANCD2-depleted cells (Figure 4C). This indicates that replication forks stall stochastically in the absence of FANCD2. Single-stranded DNA (ssDNA) gaps that accumulate behind replication forks under stressful conditions result from the degradation of nascent DNA by Mre11 (Hashimoto et al., 2010Hashimoto Y. Ray Chaudhuri A. Lopes M. Costanzo V. Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.Nat. Struct. Mol. Biol. 2010; 17: 1305-1311Crossref PubMed Scopus (358) Google Scholar). A recent study has shown that the degradation of nascent DNA was accelerated in HU-treated cancer cells lacking FANCD2 (Schlacher et al., 2012Schlacher K. Wu H. Jasin M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.Cancer Cell. 2012; 22: 106-116Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). Hence, we tested the stability of nascent DNA in primary cells deprived of nucleotides for a long period of time. HDFs were pulse labeled with CldU for 2 hr in the presence of HU and maintained in HU-containing medium for 16 hr before DNA fiber preparation. The mean length of CldU tracks in FANCD2-depleted cells was now shorter than CldU tracks from cells treated with the control siRNAs (Figures 4D and 4E and Table S3), suggesting that CldU tracts were trimmed in FANCD2-depleted cells during the 16 hr incubation period in HU. Given that uncontrolled DNA synthesis in FANCD2-depleted primary cells is likely to yield ssDNA gaps within replicated DNA molecules that are progressively degraded, these cells are expected to accumulate ssDNA. This situation echoes that of unrestrained DNA synthesis caused by Chk1 inhibition, which leads to the increased formation of ssDNA and the hyperphosphorylation of RPA32, which marks pathological DNA structures (Binz et al., 2004Binz S.K. Sheehan A.M. Wold M.S. Replication protein A phosphorylation and the cellular response to DNA damage.DNA Repair (Amst.). 2004; 3: 1015-1024Crossref PubMed Scopus (238) Google Scholar, Syljuåsen et al., 2005Syljuåsen R.G. Sørensen C.S. Hansen L.T. Fugger K. Lundin C. Johansson F. Helleday T. Sehested M. Lukas J. Bartek J. Inhibition of human Chk1 causes increase" @default.
• W1969127943 created "2016-06-24" @default.
• W1969127943 creator A5002665976 @default.
• W1969127943 creator A5016801869 @default.
• W1969127943 creator A5033324698 @default.
• W1969127943 creator A5063720817 @default.
• W1969127943 creator A5070804836 @default.
• W1969127943 creator A5082469290 @default.
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• W1969127943 date "2013-09-01" @default.
• W1969127943 modified "2023-10-15" @default.
• W1969127943 title "FANCD2 Binds MCM Proteins and Controls Replisome Function upon Activation of S Phase Checkpoint Signaling" @default.
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