Functional Interaction between the Fanconi Anemia D2 Protein and Proliferating Cell Nuclear Antigen (PCNA) via a Conserved Putative PCNA Interaction Motif

Abstract

Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway. Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway. Fanconi anemia (FA) 2The abbreviations used are: FAFanconi anemiaAPHaphidicolinFA-D2Fanconi anemia complementation group D2FANCD2Fanconi anemia D2 proteinGSTglutathione S-transferaseHRhomologous recombinationIFimmunofluorescence microscopyMMCmitomycin CPCNAproliferating cell nuclear antigenPIP-boxPCNA-interaction motifTLStranslesion DNA synthesisMOPS4-morpholinepropanesulfonic acidHAhemagglutininWCEwhole cell extractwtwild type. 2The abbreviations used are: FAFanconi anemiaAPHaphidicolinFA-D2Fanconi anemia complementation group D2FANCD2Fanconi anemia D2 proteinGSTglutathione S-transferaseHRhomologous recombinationIFimmunofluorescence microscopyMMCmitomycin CPCNAproliferating cell nuclear antigenPIP-boxPCNA-interaction motifTLStranslesion DNA synthesisMOPS4-morpholinepropanesulfonic acidHAhemagglutininWCEwhole cell extractwtwild type. is a rare recessive disorder characterized by developmental abnormalities, progressive bone marrow failure, and pronounced cancer susceptibility (1Taniguchi T. D'Andrea A.D. Blood. 2006; 107: 4223-4233Crossref PubMed Scopus (307) Google Scholar). FA patients are particularly susceptible to early-onset acute myelogenous leukemia and squamous cell carcinoma of the head, neck, and gynecologic regions (2Fanconi Anemia: Standards for Clinical Care, 2nd Ed., Fanconi Anemia Research Fund, Inc., Eugene, OR.Google Scholar). FA patient cells are hypersensitive to the clastogenic effects of DNA cross-linking agents, e.g. mitomycin C (MMC), and agents that inhibit DNA replication, e.g. aphidicolin (APH) (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar, 4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar). There are currently thirteen genetically defined FA complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, M, and N), and all thirteen genes have been identified (5Wang W. Nat. Rev. Genet. 2007; 8: 735-748Crossref PubMed Scopus (568) Google Scholar). Fanconi anemia aphidicolin Fanconi anemia complementation group D2 Fanconi anemia D2 protein glutathione S-transferase homologous recombination immunofluorescence microscopy mitomycin C proliferating cell nuclear antigen PCNA-interaction motif translesion DNA synthesis 4-morpholinepropanesulfonic acid hemagglutinin whole cell extract wild type. Fanconi anemia aphidicolin Fanconi anemia complementation group D2 Fanconi anemia D2 protein glutathione S-transferase homologous recombination immunofluorescence microscopy mitomycin C proliferating cell nuclear antigen PCNA-interaction motif translesion DNA synthesis 4-morpholinepropanesulfonic acid hemagglutinin whole cell extract wild type. A central step in the activation of the FA-BRCA pathway is the mono-ubiquitination of the FANCD2 and FANCI proteins, catalyzed by the core FA E2/E3 holoenzyme complex (5Wang W. Nat. Rev. Genet. 2007; 8: 735-748Crossref PubMed Scopus (568) Google Scholar, 6Rego M.A. Kolling 4th, F.W. Howlett N.G. Mutat. Res. 2009; 668: 27-41Crossref PubMed Scopus (19) Google Scholar). The mono-ubiquitination of FANCD2 and FANCI signals their translocation to discrete nuclear foci, where they co-localize with the BRCA1 and RAD51 DNA repair proteins, as well as the major cellular DNA polymerase processivity factor PCNA (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar, 4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar, 7Hussain S. Wilson J.B. Medhurst A.L. Hejna J. Witt E. Ananth S. Davies A. Masson J.Y. Moses R. West S.C. De Winter J.P. Ashworth A. Jones N.J. Mathew C.G. Hum. Mol. Genet. 2004; 13: 1241-1248Crossref PubMed Scopus (177) Google Scholar, 8Smogorzewska 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. Cell. 2007; 129: 289-301Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 9Taniguchi T. Garcia-Higuera I. Andreassen P.R. Gregory R.C. Grompe M. D'Andrea A.D. Blood. 2002; 100: 2414-2420Crossref PubMed Scopus (388) Google Scholar). Several studies have suggested an important role for the FA-BRCA pathway in a DNA replication-associated DNA repair process, e.g. homologous recombination (HR), and/or translesion DNA synthesis (TLS) (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar, 4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar, 10Howlett N.G. Taniguchi T. Olson S. Cox B. Waisfisz Q. De Die-Smulders C. Persky N. Grompe M. Joenje H. Pals G. Ikeda H. Fox E.A. D'Andrea A.D. Science. 2002; 297: 606-609Crossref PubMed Scopus (950) Google Scholar, 11Mirchandani K.D. McCaffrey R.M. D'Andrea A.D. DNA Repair. 2008; 7: 902-911Crossref PubMed Scopus (78) Google Scholar, 12Niedzwiedz W. Mosedale G. Johnson M. Ong C.Y. Pace P. Patel K.J. Mol. Cell. 2004; 15: 607-620Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Accordingly, additional proteins with established roles in the DNA replication stress response, including ATR, CHK1, HCLK2, and RPA, modulate DNA damage-inducible FANCD2 mono-ubiquitination (13Andreassen P.R. D'Andrea A.D. Taniguchi T. Genes Dev. 2004; 18: 1958-1963Crossref PubMed Scopus (332) Google Scholar, 14Collis S.J. Barber L.J. Clark A.J. Martin J.S. Ward J.D. Boulton S.J. Nat. Cell Biol. 2007; 9: 391-401Crossref PubMed Scopus (99) Google Scholar, 15Wang X. Kennedy R.D. Ray K. Stuckert P. Ellenberger T. D'Andrea A.D. Mol. Cell. Biol. 2007; 27: 3098-3108Crossref PubMed Scopus (120) Google Scholar). Our understanding of the regulation of this critical post-translational modification, however, is incomplete. We, and others (4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar, 7Hussain S. Wilson J.B. Medhurst A.L. Hejna J. Witt E. Ananth S. Davies A. Masson J.Y. Moses R. West S.C. De Winter J.P. Ashworth A. Jones N.J. Mathew C.G. Hum. Mol. Genet. 2004; 13: 1241-1248Crossref PubMed Scopus (177) Google Scholar) have previously reported an association between FANCD2 and PCNA. FANCD2 and PCNA co-localize in nuclear foci following treatment with agents that inhibit DNA replication. Like FANCD2, PCNA is mono-ubiquitinated following exposure to DNA-damaging agents (16Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1716) Google Scholar, 17Kannouche P.L. Wing J. Lehmann A.R. Mol. Cell. 2004; 14: 491-500Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). While FANCD2 and PCNA are mono-ubiquitinated by different E3 ubiquitin ligases, FANCL and RAD18 (16Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1716) Google Scholar, 17Kannouche P.L. Wing J. Lehmann A.R. Mol. Cell. 2004; 14: 491-500Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 18Meetei 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. Hoatlin M.E. Joenje H. Wang W. Nat. Genet. 2003; 35: 165-170Crossref PubMed Scopus (467) Google Scholar, 19Watanabe K. Tateishi S. Kawasuji M. Tsurimoto T. Inoue H. Yamaizumi M. EMBO J. 2004; 23: 3886-3896Crossref PubMed Scopus (447) Google Scholar), respectively, both proteins are de-ubiquitinated by the USP1 enzyme (20Huang T.T. Nijman S.M. Mirchandani K.D. Galardy P.J. Cohn M.A. Haas W. Gygi S.P. Ploegh H.L. Bernards R. D'Andrea A.D. Nat. Cell Biol. 2006; 8: 339-347Crossref PubMed Scopus (559) Google Scholar, 21Nijman S.M. Huang T.T. Dirac A.M. Brummelkamp T.R. Kerkhoven R.M. D'Andrea A.D. Bernards R. Mol. Cell. 2005; 17: 331-339Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). The functional significance of the FANCD2-PCNA interaction, however, has not been determined. In addition to its role as a DNA polymerase processivity factor, PCNA interacts with many DNA repair proteins, e.g. MSH3, XPG, and p21Cip1/Waf1 (22Moldovan G.L. Pfander B. Jentsch S. Cell. 2007; 129: 665-679Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar). These interactions typically occur in a hydrophobic pocket of the PCNA homotrimer, termed the interdomain connecting loop (ICL). Proteins that interact with the PCNA ICL harbor a highly conserved PCNA-binding motif called the PIP-box, defined by the amino acid sequence QXXhXXaa, where h represents amino acids with moderately hydrophobic side chains, e.g. leucine, isoleucine, or methionine (L, I, M), a represents amino acids with highly hydrophobic, aromatic side chains, e.g. phenylalanine and tyrosine (F, Y), and X is any amino acid (23Warbrick E. Bioessays. 1998; 20: 195-199Crossref PubMed Scopus (309) Google Scholar). Here, we describe an important functional interaction between FANCD2 and PCNA. We have identified a highly conserved putative PIP-box in FANCD2, and demonstrate that mutation of this motif disrupts the FANCD2-PCNA interaction, and precludes both the spontaneous and DNA damage-inducible mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant fails to correct the MMC hypersensitivity of FA-D2 patient-derived cells. However, the mutant protein retains the ability to localize to chromatin, interact with FANCE, and undergo DNA damage-inducible phosphorylation. Our results suggest that PCNA may act as a molecular platform for the mono-ubiquitination of FANCD2 and for the activation of the FA-BRCA pathway. COS-7, PD20F, and HCT116 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 12% (v/v) fetal bovine serum. PD20F pMMP-FANCD2 cells were maintained in DMEM supplemented with 1 μg/ml puromycin. PD20F plus 6xHis/V5-FANCD2 were maintained in DMEM supplemented with 200 μg/ml G418. The FANCD2 PIPΔ cDNA was generated by site-directed of the FANCD2 cDNA by site-directed and a of is a for the of C proteins. is an for were by of cells in with were in or supplemented with and B. Q. K. A. J. N. X. M. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). and of protein were used the were in (v/v) supplemented with and and with for 1 h The were as previously C. E. A.B. S. Mol. Cell. 2006; 23: Full Text Full Text PDF PubMed Scopus Google Scholar), and were with of or for 1 h Proteins were or The following were FANCD2 PCNA and and DNA polymerase FANCD2 PCNA and were and were in modified (v/v) (v/v) supplemented with with for of of protein were for 1 to were grown in in the or of MMC for with and in proteins were with a 1 1 plus for proteins, were with of for in 1 plus FANCD2 was as previously (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar, 4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar). MMC and were as previously (4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar, A. K. D'Andrea A.D. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar). of and were the for or for was the were a in the of the of we to of an association between FANCD2 and PCNA in DNA damage-inducible nuclear foci (4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar, 7Hussain S. Wilson J.B. Medhurst A.L. Hejna J. Witt E. Ananth S. Davies A. Masson J.Y. Moses R. West S.C. De Winter J.P. Ashworth A. Jones N.J. Mathew C.G. Hum. Mol. Genet. 2004; 13: 1241-1248Crossref PubMed Scopus (177) Google Scholar). were cells or with a with as well as both and mono-ubiquitinated FANCD2 we generated a FA-D2 patient cell FANCD2 of 6xHis/V5-FANCD2 the MMC hypersensitivity of cells not a acid PCNA with 6xHis/V5-FANCD2 cells the interaction between FANCD2 and PCNA in chromatin, HCT116 cells were with in and and to proteins B. Q. K. A. J. N. X. M. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). We an for mono-ubiquitinated FANCD2 in the cellular as de R. Andreassen P.R. S.P. Gregory R.C. Taniguchi T. Wang X. S. Grompe M. D'Andrea A.D. Blood. 2005; PubMed Scopus Google Scholar) and A not FANCD2 both and The of the for PCNA was by with an In a a not PCNA and HCT116 cells and In of a interaction between FANCD2 and and not to 6xHis/V5-FANCD2 in cells and this cells was used as a We identified putative PIP-box in amino acids and a amino acids The PIP-box amino acids and was for further The FANCD2 amino acids were to and the pMMP-FANCD2 PIPΔ PD20F patient cells were with pMMP-FANCD2 to the PD20F pMMP-FANCD2 PIPΔ cell the of mutation of the FANCD2 PIP-box the FANCD2-PCNA interaction, PCNA were PD20F (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar), and PIPΔ and with FANCD2 with PCNA FANCD2 with FANCD2 and of FANCD2 PIPΔ with PCNA and with a the of of PCNA in all cell cells were with 6xHis/V5-FANCD2 and PIPΔ to that 6xHis/V5-FANCD2 with PCNA 6xHis/V5-FANCD2 with 6xHis/V5-FANCD2 and of 6xHis/V5-FANCD2 PIPΔ with PCNA for 6xHis/V5-FANCD2 PIPΔ protein this to an in association between PCNA and 6xHis/V5-FANCD2 with 6xHis/V5-FANCD2 The protein has been to a molecular between the core FA complex and FANCD2, and has been to to FANCD2 P. Johnson M. Mosedale G. C. Hoatlin M. de Winter J. Joenje H. Patel K.J. EMBO J. 2002; PubMed Scopus Google Scholar). the of mutation of the FANCD2 PIP-box the interaction, we PD20F and PIPΔ cells with a and the association between FANCD2 and The of an was for this of the of a for FANCD2 protein of FANCD2 FANCD2 and FANCD2 PIPΔ with that the FANCD2 PIPΔ mutant protein retains the ability to with and We to mutation of the FANCD2 PIP-box the interaction between FANCD2 and However, we were to FANCD2 and this results suggest that the interaction between FANCD2 and PCNA is by a putative FANCD2 PIP-box that is for we the effects of mutation of the FANCD2 PIP-box FANCD2 PD20F and PIPΔ cells were to for or of FANCD2 mono-ubiquitination was for all exposure to for h the However, FANCD2 the FANCD2 PIP-box mutant protein to undergo both spontaneous or DNA damage-inducible mono-ubiquitination PCNA mono-ubiquitination was for all cell We the ability of the FANCD2 PIP-box mutant to undergo (13Andreassen P.R. D'Andrea A.D. Taniguchi T. Genes Dev. 2004; 18: 1958-1963Crossref PubMed Scopus (332) Google Scholar, S. Taniguchi T. D'Andrea A.D. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). and PD20F pMMP-FANCD2 and PIPΔ cells were in the or of treatment the of FANCD2 treatment the of both and FANCD2 PIPΔ results that the FANCD2 PIP-box mutant fails to undergo both spontaneous and DNA damage-inducible for phosphorylation. We the of the FANCD2 PIPΔ mutant were in a to extract proteins The were in a to extract nuclear proteins were with to proteins, characterized by the of the G. W. A. J.B. Johnson M. Pace P. Patel K.J. Nat. Mol. Biol. 2005; PubMed Scopus Google Scholar). We mono-ubiquitinated FANCD2 in the and in the and of as However, we both FANCD2 and FANCD2 PIPΔ in all in the and of MMC that the FANCD2 PIPΔ mutant protein for A has previously been for FANCD2 A. Mosedale G. A. Patel K.J. Mol. Cell. Biol. 2007; 27: PubMed Scopus Google Scholar). We the ability of the FANCD2 PIP-box mutant protein to discrete nuclear exposure to FANCD2 discrete nuclear foci as previously reported (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar, 4Howlett N.G. Taniguchi T. Durkin S.G. D'Andrea A.D. Glover T.W. Hum. Mol. Genet. 2005; 14: 693-701Crossref PubMed Scopus (221) Google Scholar) However, FANCD2 (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar), FANCD2 PIPΔ to nuclear foci in the or of we including nuclear as well as We the protein of FANCD2 FANCD2 and FANCD2 were to the and protein a h FANCD2 and FANCD2 PIPΔ both and FANCD2 h following exposure to FANCD2 and PIPΔ protein by and A and In protein and h following the addition of These results that the mono-ubiquitination of FANCD2 its in chromatin, and that mutation of and the PIP-box the protein We the FANCD2 PIPΔ mutant the MMC hypersensitivity of PD20F cells. In to FANCD2 the FANCD2 PIPΔ mutant to the MMC of PD20F cells. a in to the effects of MMC was for PD20F pMMP-FANCD2 PIPΔ cells with PD20F pMMP-FANCD2 cells We the of and in cells with or exposure to and MMC of in PD20F and respectively, with cells of FANCD2 the and of PD20F cells. However, of FANCD2 or FANCD2 PIPΔ to correct the of and Like PD20F a and in was for PD20F pMMP-FANCD2 PIPΔ cells following treatment with and respectively, with cells we cell in the or of Like PD20F and PD20F pMMP-FANCD2 PIPΔ cells a in of the cell following exposure to a of PD20F pMMP-FANCD2 PIPΔ cells in following exposure to with PD20F pMMP-FANCD2 cells results suggest that the FANCD2 PIP-box is for the function of FANCD2 in the cellular DNA In of an important role for the FA-BRCA pathway in a DNA repair process, both spontaneous and DNA FANCD2 mono-ubiquitination and nuclear foci occur T. Garcia-Higuera I. Andreassen P.R. Gregory R.C. Grompe M. D'Andrea A.D. Blood. 2002; 100: 2414-2420Crossref PubMed Scopus (388) Google Scholar, Olson Grompe M. Mol. Cell. Biol. 20: PubMed Scopus Google Scholar, A. Grompe M. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). it is that a or DNA replication is a major for the mono-ubiquitination of FANCD2. The by FANCD2 is to damaged DNA replication has however, been determined. Here, we describe an important interaction between the FANCD2 and PCNA proteins. We demonstrate that FANCD2 and PCNA in chromatin, and that this association is a highly conserved FANCD2 PIP-box of this motif both spontaneous and DNA damage-inducible FANCD2 Our results suggest that the association between PCNA and FANCD2 may an important for the mono-ubiquitination of FANCD2 its DNA repair A role for via PIP-box has been established for DNA repair including MSH3, XPG, and N. H. I. Johnson J. S. 2008; PubMed Scopus Google Scholar, R. H.L. M.A. M.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Johnson I. B. J. S. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, G. T. J. Genes Dev. 2001; 15: PubMed Scopus Google Scholar). The association of FANCD2 with PCNA in a DNA represents an for the of FANCD2 to of DNA by the DNA the mutation of the FANCD2 PIP-box both spontaneous and DNA damage-inducible FANCD2 mono-ubiquitination was FANCD2 that of the of ubiquitin has been that to a of FANCD2 mono-ubiquitination (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar). We not the that a in protein the of the FANCD2 PIP-box mutant to undergo as the PIP-box is to (3Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar). a in protein may of the FA E2/E3 holoenzyme to Several of however, a FANCD2 the FANCD2 PIPΔ mutant the ability to interact with FANCE, a core FA complex protein previously to to FANCD2 P. Johnson M. Mosedale G. C. Hoatlin M. de Winter J. Joenje H. Patel K.J. EMBO J. 2002; PubMed Scopus Google Scholar). the FANCD2 PIPΔ mutant protein DNA damage-inducible (13Andreassen P.R. D'Andrea A.D. Taniguchi T. Genes Dev. 2004; 18: 1958-1963Crossref PubMed Scopus (332) Google Scholar, S. Taniguchi T. D'Andrea A.D. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). the FANCD2 PIPΔ mutant protein to These results suggest that the mutant protein retains and we the of a or that mutation of the PIP-box precludes the interaction of FANCD2 with a protein, for its The mono-ubiquitination of FANCD2 has previously been to for its targeting to de R. Andreassen P.R. S.P. Gregory R.C. Taniguchi T. Wang X. S. Grompe M. D'Andrea A.D. Blood. 2005; PubMed Scopus Google Scholar). However, in this study, a different we FANCD2 and FANCD2 PIPΔ proteins in We that mono-ubiquitination FANCD2 in chromatin, and that FANCD2 fails to in In of a FANCD2 mono-ubiquitination the FANCD2 enzyme A.M. D'Andrea A.D. A. Mol. Cell. 2006; 23: Full Text Full Text PDF PubMed Scopus Google Scholar) has been to of DNA or cell A. Mosedale G. A. Patel K.J. Mol. Cell. Biol. 2007; 27: PubMed Scopus Google Scholar). the FA core complex to chromatin, and its E3 ubiquitin is its DNA A. Mosedale G. A. Patel K.J. Mol. Cell. Biol. 2007; 27: PubMed Scopus Google Scholar, J. Blood. 2005; PubMed Scopus Google Scholar). we that the association between FANCD2 and PCNA may facilitate the FA E2/E3 mono-ubiquitination of FANCD2, and its and in and H.J. Pace P. W. L.J. M. M. Patel K.J. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). with we have that mono-ubiquitinated FANCD2 a protein FANCD2 in FANCD2 may as a molecular between FANCD2 and an as In of this N. H. M. N. S. K. T. P.J. A.R. H. M. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) that a protein an mutation in the hydrophobic of ubiquitin disrupts the of cells. Several ubiquitin including or of the core FA complex proteins. important FANCD2 function for the FA core complex in has been K.D. McCaffrey R.M. D'Andrea A.D. DNA Repair. 2008; 7: 902-911Crossref PubMed Scopus (78) Google and not FANCD2, were to for the of the protein nuclear foci K.D. McCaffrey R.M. D'Andrea A.D. DNA Repair. 2008; 7: 902-911Crossref PubMed Scopus (78) Google Scholar). has been that the mono-ubiquitination of PCNA may for a DNA repair process, for repair W. Mosedale G. Johnson M. Ong C.Y. Pace P. Patel K.J. Mol. Cell. 2004; 15: 607-620Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, C.E. L.J. Mol. Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, G. R. DNA Repair. 2007; PubMed Scopus Google Scholar). repair is to both and a W. Mosedale G. Johnson M. Ong C.Y. Pace P. Patel K.J. Mol. Cell. 2004; 15: 607-620Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, G. R. DNA Repair. 2007; PubMed Scopus Google Scholar), we a FANCD2 and a process, may function in the repair of is with studies a role for the FA proteins in an process, as well as that the major proteins FANCD1/BRCA2, and RAD51, co-localize with PCNA and FANCD2 in nuclear foci J. G. T. R. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. K. M. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In suggest that the FANCD2 protein with PCNA in a DNA via a highly conserved of a DNA replication, FANCD2 mono-ubiquitinated via the of the core FA the FANCL ubiquitin and the its function in DNA The or repair of damaged DNA replication to the of the in FA cancer cells. Our have for understanding of the DNA and repair DNA replication and further the molecular of a highly cancer susceptibility We of the Howlett and Glover R. Martin and for We K. J. Patel for the with