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DNA Damage and Cellular Stress Responses

Participation of kin17 Protein in Replication Factories and in Other DNA Transactions Mediated by High Molecular Weight Nuclear Complexes¹

¹ CEA, Commissariat à l'Energie Atomique, Laboratoire de Génétique de la Radiosensibilité, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, Fontenay-aux-Roses, France;
² CNRS, Institut André Lwoff, FR2249, UPR 1983, Génétique moléculaire et intégration des fonctions cellulaires, Villejuif Cedex, France; and
³ CEA, Commissariat à l'Energie Atomique, Service de Pharmacologie et d'Immunologie, Département de Recherche Médicale, Direction des Sciences du Vivant, CE Saclay, Gif-sur-Yvette, France

Requests for reprints: Denis S.F. Biard, CEA-DSV-DRR, Laboratoire de Génétique de la Radiosensibilité, BP 6, 92265 Fontenay aux Roses, France. E-mail: biard{at}dsvidf.cea.fr

The Homo sapiens kin17 (_HSAkin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous _HSAkin17 protein revealed an ultrastructural co-localization of _HSAkin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that _HSAkin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of _HSAkin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound _HSAkin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced _HSAkin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous _HSAkin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M_r 600,000. Interestingly, HU-induced G₁-S arrest triggered an increase in the molecular weight of complexes containing _HSAkin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound _HSAkin17 protein. We hypothesize that in the presence of unrepaired DNA damage, _HSAkin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.

Key Words: kin17 • DNA replication • DNA damage • L-mimosine • hydroxyurea

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