RESUMO
Puralpha is a sequence-specific single-stranded nucleic acid-binding protein and a member of the highly conserved Pur family. Puralpha has been shown to colocalize with cyclin A/Cdk2 and to coimmunoprecipitate with cyclin A during S-phase. Here we show that this interaction is mediated by a specific affinity of Puralpha for Cdk2. In pull-down assays GST-Puralpha efficiently binds Cdk2 and Cdk1, binds Cdk4 less efficiently, and does not display binding to Cdk6. Puralpha stimulates several-fold the phosphorylation in vitro of histone H1 by cyclin A/Cdk2, produced from baculovirus constructs. Double chromatin immunoprecipitation using antibodies to Cdk2 and Puralpha reveals that both proteins colocalize in HeLa cells to DNA segments upstream of the c-MYC gene. Pur family member Purgamma colocalizes with Cdk2 to a specific DNA segment in this region.
Assuntos
Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/química , Ciclina A/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Quinase 2 Dependente de Ciclina , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Ligação ProteicaRESUMO
Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.
