RESUMO
The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia telangiectasia mutated). We reveal a function for MRN in ATR (ATM- and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required for recruitment of TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction and that this recruitment is necessary for phosphorylation of CHK1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at an ssDNA-to-dsDNA junction, 911 is required for TOPBP1 "activation." These findings provide molecular insights into how ATR is activated.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Proteínas Cromossômicas não Histona/metabolismo , Enzimas Reparadoras do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Proteína Homóloga a MRE11 , Complexos Multiproteicos , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , MicroRNAs/genética , Processamento Alternativo , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Transfecção , DNA Metiltransferase 3BRESUMO
Cyclin-dependent kinases (CDKs) play a crucial role in cell cycle progression by controlling the transition from G(1) phase into S phase where DNA is replicated. Key to this transition is the regulation of initiation of DNA replication at replication origins. CDKs are thought to regulate origins of replication both positively and negatively by phosphorylating replication proteins at origins. Several replication proteins that are potentially negatively regulated upon CDK phosphorylation have been identified. However, the mechanism by which CDKs activate replication is currently less well understood. New observations revealing that the initiation protein Cdc6 is stabilized by CDK2-dependent phosphorylation may give more insight in this process.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , Modelos Genéticos , Complexo de Reconhecimento de Origem/genéticaRESUMO
Activating point mutations of the small GTPase Ras are present in about 30% of all human tumors. Constitutively active Ras induces growth factor independent cell proliferation and cell survival. Oncogenic Ras appears to be essential for tumor progression and maintenance. Several therapeutic agents have been developed to inhibit Ras, such as FTIs and antisense oligonucleotides. A new tool for blocking oncogenes in cancer cells has emerged with the discovery that RNA interference can specifically silence expression of endogenous human genes. The therapeutic potential of a RNAi-mediating vector was recently demonstrated by the stable suppression of oncogenic K-Ras in tumor cells.