ABSTRACT
We describe the cloning and characterization of the human KIN17 cDNA encoding a 45 kDa zinc finger nuclear protein. Previous reports indicated that mouse kin17 protein may play a role in illegitimate recombination and in gene regulation. Furthermore, overproduction of mouse kin17 protein inhibits the growth of mammalian cells, particularly the proliferation of human tumour-derived cells. We show here that the KIN17 gene is remarkably conserved during evolution. Indeed, the human and mouse kin17 proteins are 92.4% identical. Furthermore, DNA sequences from fruit fly and filaria code for proteins that are 60% identical to the mammalian kin17 proteins, indicating conservation of the KIN17 gene among metazoans. The human KIN17 gene, named (HSA)KIN17, is located on human chromosome 10 at p15-p14. The (HSA)KIN17 RNA is ubiquitously expressed in all the tissues and organs examined, although muscle, heart and testis display the highest levels. UVC irradiation of quiescent human primary fibroblasts increases (HSA)KIN17 RNA with kinetics similar to those observed in mouse cells, suggesting that up-regulation of the (HSA)KIN17 gene after UVC irradiation is a conserved response in mammalian cells. (HSA)kin17 protein is concentrated in intranuclear focal structures in proliferating cells as judged by indirect immunofluorescence. UVC irradiation disassembles (HSA)kin17 foci in cycling cells, indicating a link between the intranuclear distribution of (HSA)kin17 protein and the DNA damage response.
Subject(s)
DNA, Complementary/genetics , DNA, Complementary/radiation effects , DNA-Binding Proteins/genetics , Nuclear Proteins , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Base Sequence , Brugia malayi/genetics , Cell Division/physiology , Cell Nucleus/metabolism , Cells, Cultured , Chromosomes, Human, Pair 10/genetics , Cloning, Molecular , Conserved Sequence , DNA Damage , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Drosophila melanogaster/genetics , Evolution, Molecular , Fibroblasts/metabolism , Fibroblasts/physiology , Fibroblasts/radiation effects , Gene Expression , Humans , Mice , Molecular Sequence Data , RNA/genetics , RNA/metabolism , RNA/radiation effects , RNA-Binding Proteins , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Ultraviolet Rays , Zinc Fingers/radiation effectsABSTRACT
To characterize the biological role of Kin17 protein, a mammalian nuclear protein which participates in the response to UV and ionizing radiation and binds to curved DNA, EBV-derived vectors carrying (Mm)Kin17 cDNA were constructed and transfected in tumorigenic cells harboring different p53 profiles (HeLa, H1299, and HCT116) and in immortalized HEK 293 cells. (Mm)Kin17 protein expression induced a tremendous decrease in cell proliferation of the three tumorigenic cell lines 2 weeks after transfection. Transfection of HEK 293 cells with an pEBVCMV(Mm)Kin17 plasmid gave rise to numerous (Mm)Kin17-expressing cells which constantly disappeared with time, preventing the establishment of (Mm)Kin17-expressing cells. Several independent clones were isolated from HEK 293 cells carrying a pEBVMT(Mm)Kin17 vector. The two clones described here (B223.1 and B223.2) exhibited different (Mm)Kin17 protein levels and displayed a gradual decrease in their proliferative capacities. In B223.1 cells, the basal expression of (Mm)Kin17 greatly reduced plating efficiency and cell growth. B223.1 cell morphology was altered, with numerous round-shaped cells whose spreading on the culture support was hampered. We observed giant multinucleated cells or cells containing micronuclei-like structures and/or multilobed nuclei. To conclude, (Mm)Kin17 overexpression reduced the proliferation of tumorigenic cells independently of their p53 status and modified cell growth and cell morphology of established HEK 293 cells producing (Mm)Kin17 protein. It is likely that (Mm)Kin17 may interfere with DNA replication.
Subject(s)
Cell Division , DNA-Binding Proteins/physiology , Nuclear Proteins , Animals , Blotting, Western , Cadmium/pharmacology , Cell Count , Cell Line, Transformed , Cell Size , Clone Cells/cytology , Clone Cells/drug effects , Clone Cells/metabolism , Cyclin B/metabolism , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique, Indirect , Gene Expression/drug effects , Giant Cells/metabolism , Humans , In Situ Hybridization, Fluorescence , Mice , Proliferating Cell Nuclear Antigen/metabolism , RNA-Binding Proteins , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , Zinc/pharmacologyABSTRACT
We used antibodies against E coli RecA protein to identify in mouse cells a 45-kDa DNA-binding protein called kin17, which has an active zinc finger and a nuclear localisation signal. Kin17 protein produced in E coli binds preferentially to the curved DNA of a bacterial promoter in vivo and in vitro, suggesting a transcriptional regulation activity. The fact that in rodent cells kin17 protein levels increase after gamma-irradiation suggests its participation in a cellular response to ionising radiation. We raised polyclonal antibodies against the whole kin17 protein and against its derived synthetic peptides. We report the detection of kin17 protein and of truncated forms of the protein by Western blot or by immunocytochemistry after transient overexpression in cultured human cells. Our results indicate that the cross-reactivity with the anti-RecA antibodies is due to an antigenic determinant located in the core of kin17 protein, between residues 129 and 228. The kin17 protein is located in the nucleus and is concentrated in small nuclear dot-like structures throughout the nucleoplasm. The RecA homologous region seems to play an essential role in the localisation of kin17 protein since the deletion of this particular region dramatically changes the form and the distribution of the intranuclear foci. We hypothesise that these dot-like structures reflect nuclear metabolism compartmentalization.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins/biosynthesis , Nuclear Proteins , Animals , Blotting, Western , Cell Nucleus/genetics , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Mice , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Nuclear Localization Signals/genetics , Protein Structure, Tertiary , RNA-Binding Proteins , Rec A Recombinases/genetics , Sequence Deletion , Subcellular Fractions/metabolism , Transfection , Zinc Fingers/geneticsABSTRACT
We report the isolation of the mouse Kin17 gene, located on chromosome 2, coding a nuclear Zn-finger protein that has a 39-residue region homologous to Escherichia coli RecA protein and that is recognized by anti-RecA antibodies. Kin17 protein preferentially binds to curved DNA in vitro and in vivo, suggesting a role in illegitimate recombination and in regulation of gene expression. We have shown that the Kin17 gene is about 8 kb in length and displays three exons and two introns. The 5' flanking region lacks a canonical TATAA box but presents several putative regulatory domains. A major transcription initiation site is located 322 nucleotides upstream of the translation start site. The 1.7-kb transcript of the Kin17 gene is weakly and ubiquitously expressed in murine tissues and cell lines as determined by Northern analysis. The cross-hybridization of Kin17 cDNA with the genomic DNA of other species in Southern analysis indicates the conservation of the gene among mammals and suggests that the Kin17 gene plays a conserved role in DNA metabolism.
Subject(s)
DNA-Binding Proteins/genetics , Nuclear Proteins , Zinc Fingers/genetics , Animals , Base Sequence , Cattle , Chickens , Cloning, Molecular , DNA , Dogs , Humans , In Situ Hybridization , Introns , Macaca mulatta , Mice , Mice, Inbred BALB C , Molecular Sequence Data , RNA-Binding Proteins , Rabbits , Rats , Rats, Sprague-DawleyABSTRACT
We have sought to characterize the molecular basis of the sensitivity to ionising radiation and to identify the genes involved in the cellular response of mammalian cells to such radiation. Using the Escherichia coli model, we tested the hypothesis that functional domains of RecA protein are represented in proteins of mammalian cells. We review here the results obtained in the detection of nuclear proteins of mammalian cells that are recognized by anti-RecA antibodies. We have called them kin proteins. Kin proteins likely play a role in DNA metabolism. We summarize the cloning of the mouse Kin-17 cDNA and our work on the identification and preliminary characterisation of the biochemical properties of mouse kin17 protein, a new nuclear protein able to recognize bent DNA and suspected to be involved in illegitimate recombination. We briefly describe our latest experiments on the molecular characterisation of the mouse Kin-17 gene. Finally, we discuss the properties of kin17 protein and the possible participation of kin17 protein in DNA transactions like transcription or recombination.
