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Overexpression of kin17 protein forms intranuclear foci in mammalian cells
P Kannouche1, G Pinon-Lataillade, P Mauffrey
1Département de Radiobiologie et de Radiopathologie, Centre d'Etudes de Fontenay-aux-Roses, CEA, France.
Abstract:
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.
Insights
Researchers identified kin17, a DNA-binding protein involved in cellular responses to DNA damage and radiation. Kin17
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- A 45-kDa DNA-binding protein, kin17, was identified in mouse cells using antibodies against E. coli RecA protein.
- Kin17 possesses an active zinc finger and a nuclear localization signal, suggesting roles in nuclear processes.
- Kin17 protein levels increase in rodent cells following gamma-irradiation, indicating a role in DNA damage response.
Purpose of the Study:
- To characterize the kin17 protein, its function, and its cellular localization.
- To investigate the interaction of kin17 with DNA and its potential role in transcriptional regulation.
- To elucidate the role of the RecA homologous region in kin17's nuclear localization and function.
Main Methods:
- Antibodies against E. coli RecA protein were used for initial identification of kin17.
- Kin17 protein produced in E. coli was used for in vitro and in vivo DNA-binding assays.
- Polyclonal antibodies against kin17 were generated for detection via Western blot and immunocytochemistry.
- Analysis of truncated kin17 forms to determine the role of the RecA homologous region in localization.
Main Results:
- Kin17 binds preferentially to curved DNA, specifically bacterial promoter DNA, suggesting transcriptional regulatory activity.
- Kin17 protein levels elevate in rodent cells post-gamma irradiation, implicating it in the cellular response to ionizing radiation.
- Cross-reactivity with anti-RecA antibodies is attributed to an antigenic determinant within kin17's core (residues 129-228).
- Kin17 localizes to the nucleus, forming distinct dot-like structures; deletion of the RecA homologous region alters these structures' morphology and distribution.
Conclusions:
- Kin17 is a nuclear DNA-binding protein with potential transcriptional regulatory functions.
- Kin17 is involved in the cellular response to DNA damage, particularly ionizing radiation.
- The RecA homologous region is crucial for kin17's nuclear localization and the formation of intranuclear foci, potentially reflecting nuclear compartmentalization.