title: Coordinate nuclear targeting of the FANCD2 and FANCI proteins via a FANCD2 nuclear localization signal.
abstract: Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. 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. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island, United States of America.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Boisvert
authors.forename: Meghan A
authors.initials: MA
authors.lastname: Rego
authors.forename: Paul A
authors.initials: PA
authors.lastname: Azzinaro
authors.forename: Maurizio
authors.initials: M
authors.lastname: Mauro
authors.forename: Niall G
authors.initials: NG
authors.lastname: Howlett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D017124:Conserved Sequence; D004249:DNA Damage; D005199:Fanconi Anemia / Q000235:genetics / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics; D021381:Protein Transport; D016415:Sequence Alignment; D054875:Ubiquitination
chemical_list: D002843:Chromatin; C519921:FANCI protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; D019913:Nuclear Localization Signals