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Human kin17 protein directly interacts with the simian virus 40 large T antigen and inhibits DNA replication
Laurent Miccoli1, Denis S F Biard, Christophe Créminon
1CEA, Commissariat à l'Energie Atomique, Laboratoire de Génétique de la Radiosensibilité, Direction des Sciences du Vivant, 92265 Fontenay-aux-Roses, France. miccoli@dsvidf.cea.fr
Abstract:
Kin17 is an evolutionarily conserved DNA-binding protein, which forms intranuclear foci in proliferating cells. Recent data have suggested that human kin17 protein is associated with cell proliferation and unrepaired DNA lesions. Herein, we show that human fibroblasts (MRC5-V2 and CHSV4) immortalized with SV40 overexpress endogenous kin17 protein, as compared with normal diploid human fibroblasts. We observed that certain carcinoma cell lines also up-regulated kin17 protein, suggesting that increased kin17 protein levels may be a consequence of the immortalized phenotype. We report here that the endogenous kin17 protein is located in nucleoplasmic foci and colocalizes with SV40 large T antigen. Purification of human kin17 protein allowed analysis of the physical interaction with T antigen by several in vitro and in vivo assays. Large T antigen and human kin17 protein are part of the same high molecular weight multiprotein complex in human cells. Furthermore, human kin17 protein interacts with T antigen bound to the SV40 DNA origin of replication. Strikingly, the overexpression of human kin17 protein in vivo and the introduction of increased amounts of human kin17 protein in an in vitro assay reduced T-antigen-dependent DNA replication, suggesting that kin17 protein may be involved in the DNA replication process in human cells.
Insights
Kin17 protein, linked to cell proliferation, interacts with SV40 T antigen. Overexpression of Kin17 inhibits T-antigen-dependent DNA replication, suggesting its role in human DNA replication.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Kin17 is an evolutionarily conserved DNA-binding protein found in intranuclear foci of proliferating cells.
- Previous studies suggest human kin17 protein involvement in cell proliferation and unrepaired DNA lesions.
Purpose of the Study:
- To investigate the role of human kin17 protein in cell proliferation and its interaction with SV40 large T antigen.
- To determine if kin17 protein levels are altered in immortalized cells and carcinomas.
Main Methods:
- Comparative analysis of kin17 protein expression in normal, SV40-immortalized human fibroblasts, and carcinoma cell lines.
- Immunofluorescence to assess kin17 localization and colocalization with SV40 T antigen.
- In vitro and in vivo assays to study the physical interaction between human kin17 and T antigen.
Main Results:
- SV40-immortalized fibroblasts and some carcinoma cell lines showed overexpression of endogenous kin17 protein.
- Endogenous kin17 protein localized to nucleoplasmic foci and colocalized with SV40 T antigen.
- Human kin17 protein and T antigen form a multiprotein complex and interact at the SV40 DNA origin of replication.
Conclusions:
- Increased kin17 protein levels may correlate with the immortalized cell phenotype.
- Kin17 protein interacts with SV40 T antigen and is part of a multiprotein complex.
- Kin17 protein negatively regulates T-antigen-dependent DNA replication, indicating a potential role in human DNA replication processes.