RESUMO
Mammalian DNA replication origins are "licensed" by the loading of DNA helicases, a reaction that is mediated by CDC6 and CDT1 proteins. After initiation of DNA synthesis, CDC6 and CDT1 are inhibited to prevent origin reactivation and DNA overreplication before cell division. CDC6 and CDT1 are highly expressed in many types of cancer cells, but the impact of their deregulated expression had not been investigated in vivo. Here, we have generated mice strains that allow the conditional overexpression of both proteins. Adult mice were unharmed by the individual overexpression of either CDC6 or CDT1, but their combined deregulation led to DNA re-replication in progenitor cells and lethal tissue dysplasias. This study offers mechanistic insights into the necessary cooperation between CDC6 and CDT1 for facilitation of origin reactivation and describes the physiological consequences of DNA overreplication.
Assuntos
Replicação do DNA , Diarreia Infantil/genética , Mucosa Intestinal/metabolismo , Síndromes de Malabsorção/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diarreia Infantil/metabolismo , Feminino , Mucosa Intestinal/patologia , Síndromes de Malabsorção/metabolismo , Masculino , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , TransgenesRESUMO
Cdc6 encodes a key protein for DNA replication, responsible for the recruitment of the MCM helicase to replication origins during the G1 phase of the cell division cycle. The oncogenic potential of deregulated Cdc6 expression has been inferred from cellular studies, but no mouse models have been described to study its effects in mammalian tissues. Here we report the generation of K5-Cdc6, a transgenic mouse strain in which Cdc6 expression is deregulated in tissues with stratified epithelia. Higher levels of CDC6 protein enhanced the loading of MCM complexes to DNA in epidermal keratinocytes, without affecting their proliferation rate or inducing DNA damage. While Cdc6 overexpression did not promote skin tumors, it facilitated the formation of papillomas in cooperation with mutagenic agents such as DMBA. In addition, the elevated levels of CDC6 protein in the skin extended the resting stage of the hair growth cycle, leading to better fur preservation in older mice.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cabelo/metabolismo , Proteínas Nucleares/metabolismo , Papiloma/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Fragmentação do DNA , Replicação do DNA/genética , Replicação do DNA/fisiologia , Feminino , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Papiloma/genética , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC.