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Characterization of a Putative Nuclear Localization Signal in the Fanconi Anemia D2 Protein
Author(s) -
Boisvert Rebecca Anne,
Rego Meghan A,
Mauro Maurizio,
Howlett Niall G
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.758.1
Subject(s) - fancd2 , nls , nuclear localization sequence , chromatin , biology , fanconi anemia , mutant , microbiology and biotechnology , dna repair , genetics , gene
Fanconi anemia (FA) is a rare disease characterized by bone marrow failure and increased cancer susceptibility. FA is caused by mutation of any one of 15 known genes. The FA proteins function collectively to repair DNA interstrand cross‐links (ICLs) and to prevent cellular transformation. Following exposure to ICLinducing agents, e.g. mitomycin C (MMC), the FA core complex catalyzes the mono‐ubiquitination of the FANCD2 and FANCI proteins, targeting them for chromatin localization. Currently, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, and, in particular, how these proteins are recruited to chromatin. In this study, using an in sili c o approach, we have identified a highly conserved bipartite nuclear localization signal (NLS) in the amino‐terminus of FANCD2. When fused to the coding sequence of GFP, the FANCD2 NLS promoted exclusive nuclear expression of GFP. Using deletion and site‐directed mutagenesis strategies, we have generated FANCD2 mutants lacking 57 (ΔN57) or 100 (ΔN100) amino‐terminal residues, and a missense mutant changing amino acids K4, R5, R6 to N4, N5, N6 respectively (3N). Our preliminary studies demonstrate that the NLS is required for FANCD2 and FANCI monoubiquitination, and their localization to and retention in chromatin. Consequently, the NLS mutants fail to rescue the MMC hypersensitivity of FA‐D2 ( FANCD2−/− ) cells. Our results corroborate our hypothesis that FANCD2 and FANCI enter the nucleus as a heterodimer primarily via the FANCD2 NLS, lending important insight into the regulation of a major tumor suppressor network.