RESUMO
The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Heterocromatina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Homólogo 5 da Proteína Cromobox/genética , Homólogo 5 da Proteína Cromobox/metabolismo , Instabilidade Cromossômica , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Heterocromatina/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Transdução de SinaisRESUMO
Polo-like kinase 1 (PLK1) plays key roles during mitosis, prompting the development of PLK1 inhibitors for anticancer therapy. We recently determined that PLK1 is crucially required for entry into mitosis. Hence, we discuss the potential and limitations of PLK1 inhibition strategies to promote mitotic arrest and death of cancer cells.
RESUMO
The regulation of proteolysis is an efficient way to control protein function in cells. Here, we present a general strategy enabling to increase the spatiotemporal resolution of conditional proteolysis by using light activation as trigger. Our approach relies on the auxin-inducible degradation system obtained by transposing components of the plant auxin-dependent degradation pathway in mammalian cells. We developed a photoactivatable auxin that acts as a photoactivatable inducer of degradation. Upon local and short light illumination, auxin is released in cells and triggers the degradation of a protein of interest with spatiotemporal control.
