title: Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair.
abstract: Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.
authors.affiliation: Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: J
authors.initials: J
authors.lastname: Qiu
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: G
authors.initials: G
authors.lastname: Frank
authors.forename: B
authors.initials: B
authors.lastname: Shen
mesh_terms: D002453:Cell Cycle; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D045585:Flap Endonucleases; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D019913:Nuclear Localization Signals; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D021381:Protein Transport; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D014170:Transformation, Genetic
chemical_list: D019913:Nuclear Localization Signals; D018809:Proliferating Cell Nuclear Antigen; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human