Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes.
The Homo sapiens kin17 ((HSA)kin17) 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 (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 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 (HSA)kin17 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 (HSA)kin17 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 (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 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 (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.
Biard
Denis S F
DS
CEA, Commissariat à l'Energie Atomique, Laboratoire de Génétique de la Radiosensibilité, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, BP 6, 92265 Fontenay-aux-Roses, France. biard@dsvidf.cea.fr
Miccoli
Laurent
L
Despras
Emmanuelle
E
Harper
Francis
F
Pichard
Evelyne
E
Créminon
Christophe
C
Angulo
Jaime F
JF
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D001973:Bromodeoxyuridine; D002289:Carcinoma, Non-Small-Cell Lung; D002455:Cell Division; D003110:Colonic Neoplasms; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D006918:Hydroxyurea / Q000633:toxicity; D008175:Lung Neoplasms; D016253:Microscopy, Immunoelectron; D008898:Mimosine / Q000633:toxicity; D009687:Nuclear Proteins / Q000032:analysis / Q000502:physiology*; D016601:RNA-Binding Proteins; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; C489487:KIN protein, human; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D008898:Mimosine; D001973:Bromodeoxyuridine; D006918:Hydroxyurea