RESUMO
The DNA damage sensor Mre11-Rad50-Nbs1 complex and Polo kinase are recruited to DNA lesions during mitosis. However, their mechanism of recruitment is elusive. Here, using live-cell imaging combined with micro-irradiation of single chromosomes, we analyze the dynamics of Polo and Mre11 at DNA lesions during mitosis in Drosophila These two proteins display distinct kinetics. Whereas Polo kinetics at double-strand breaks (DSBs) are Cdk1-driven, Mre11 promptly but briefly associates with DSBs regardless of the phase of mitosis and re-associates with DSBs in the proceeding interphase. Mechanistically, Polo kinase activity is required for its own recruitment and that of the mitotic proteins BubR1 and Bub3 to DSBs. Moreover, depletion of Rad50 severely impaired Polo kinetics at mitotic DSBs. Conversely, ectopic tethering of Mre11 to chromatin was sufficient to recruit Polo. Our study highlights a novel pathway that links the DSB sensor Mre11-Rad50-Nbs1 complex and Polo kinase to initiate a prompt, decisive response to the presence of DNA damage during mitosis.
Assuntos
Proteínas de Drosophila , Drosophila , Hidrolases Anidrido Ácido , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases , Proteína Homóloga a MRE11/genética , Mitose/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Chromatid segregation must be coordinated with cytokinesis to preserve genomic stability. Here we report that cells clear trailing chromatids from the cleavage site by undergoing two phases of cell elongation. The first phase relies on the assembly of a wide contractile ring. The second phase requires the activity of a pool of myosin that flows from the ring and enriches the nascent daughter cell cortices. This myosin efflux is a novel feature of cytokinesis and its duration is coupled to nuclear envelope reassembly and the nuclear sequestration of the Rho-GEF Pebble. Trailing chromatids induce a delay in nuclear envelope reassembly concomitant with prolonged cortical myosin activity, thus providing forces for the second elongation. We propose that the modulation of cortical myosin dynamics is part of the cellular response triggered by a "chromatid separation checkpoint" that delays nuclear envelope reassembly and, consequently, Pebble nuclear sequestration when trailing chromatids are present at the midzone.Chromatid segregation must be coordinated with cytokinesis to preserve genomic stability. Here the authors show that cells clear trailing chromatids from the cleavage site in a two-step cell elongation and demonstrate the role of myosin efflux in the second phase.
Assuntos
Processos de Crescimento Celular/genética , Segregação de Cromossomos/genética , Citocinese/genética , Miosinas/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Cromátides/genética , Cromátides/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Miosinas/metabolismo , Transporte Proteico , Pupa/citologia , Pupa/genética , Pupa/metabolismo , Imagem com Lapso de Tempo/métodosRESUMO
The presence of DNA double-strand breaks during mitosis is particularly challenging for the cell, as it produces broken chromosomes lacking a centromere. This situation can cause genomic instability resulting from improper segregation of the broken fragments into daughter cells. We recently uncovered a process by which broken chromosomes are faithfully transmitted via the BubR1-dependent tethering of the two broken chromosome ends. However, the mechanisms underlying BubR1 recruitment and function on broken chromosomes were largely unknown. We show that BubR1 requires interaction with Bub3 to localize on the broken chromosome fragments and to mediate their proper segregation. We also find that Cdc20, a cofactor of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), accumulates on DNA breaks in a BubR1 KEN box-dependent manner. A biosensor for APC/C activity demonstrates a BubR1-dependent local inhibition of APC/C around the segregating broken chromosome. We therefore propose that the Bub3-BubR1 complex on broken DNA inhibits the APC/C locally via the sequestration of Cdc20, thus promoting proper transmission of broken chromosomes.
