RESUMO
Levels of residual structure in disordered interaction domains determine in vitro binding affinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage. These results establish that residual structure is an important determinant of signaling fidelity in cells.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Proto-Oncogênicas c-mdm2/química , Transdução de Sinais , Proteína Supressora de Tumor p53/química , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Dano ao DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Raios gama , Regulação da Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Transposons are natural gene delivery vehicles. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in the cells of vertebrates including humans. SB transposition into chromosomal DNA occurs in a fairly random manner. This is clearly not desirable in human gene therapeutic applications because there are potential genotoxic effects associated with transposon integration. In this study we set out to manipulate the selection of SB's target sites for targeted transposition into predetermined chromosomal regions. We evaluated experimental strategies based on engineered proteins composed of DNA-binding domains fused to (i) the transposase; (ii) another protein that binds to a specific DNA sequence within the transposable element; and (iii) another protein that interacts with the transposase. We demonstrated targeted transposition into endogenous matrix attachment regions (MARs) and a chromosomally integrated tetracycline response element (TRE) in cultured human cells, using targeting proteins that bind to the transposon DNA. An approach based on interactions between the transposase and a targeting protein containing the N-terminal protein interaction domain of SB was found to enable an approximately 10(7)-fold enrichment of transgene insertion at a desired locus. Our experiments provide proof-of-principle for targeted chromosomal transposition of an otherwise randomly integrating transposon. Targeted transposition can be a powerful technology for safe transgene integration in human therapeutic applications.
Assuntos
Elementos de DNA Transponíveis/genética , Genoma Humano/genética , Mutagênese Insercional , Sequência de Bases , Sítios de Ligação/genética , DNA/genética , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transposases/genética , Transposases/metabolismoRESUMO
We have developed a stable RNA interference (RNAi) delivery system that is based on the Frog Prince transposable element. This plasmid-based vector system combines the gene silencing capabilities of H1 polymerase III promoter-driven short hairpin RNAs (shRNA) with the advantages of stable and efficient genomic integration of the shRNA cassette mediated by transposition. We show that the Frog Prince-based shRNA expressing system can efficiently knock down the expression of both exogenous as well as endogenous genes in human cells. Furthermore, we use the Frog Prince-based system to study the effect of knockdown of the DNA repair factor Ku70 on transposition of the Sleeping Beauty transposon. Transposon-mediated genomic integration ensures that the shRNA expression cassette and a selectable marker gene within the transposon remain intact and physically linked. We demonstrate that a major advantage of our vector system over plasmid-based shRNA delivery is both its enhanced frequency of intact genomic integration as well as higher target suppression in transgenic human cells. Due to its simplicity and effectiveness, transposon-based RNAi is an emerging tool to facilitate analysis of gene function through the establishment of stable loss-of-function cell lines.
RESUMO
The Sleeping Beauty (SB) element is a useful tool to probe transposon-host interactions in vertebrates. We investigated requirements of DNA repair factors for SB transposition in mammalian cells. Factors of nonhomologous end joining (NHEJ), including Ku, DNA-PKcs, and Xrcc4 as well as Xrcc3/Rad51C, a complex that functions during homologous recombination, are required for efficient transposition. NHEJ plays a dominant role in repair of transposon excision sites in somatic cells. Artemis is dispensable for transposition, consistent with the lack of a hairpin structure at excision sites. Ku physically interacts with the SB transposase. DNA-PKcs is a limiting factor for transposition and, in addition to repair, has a function in transposition that is independent from its kinase activity. ATM is involved in excision site repair and affects transposition rates. The overlapping but distinct roles of repair factors in transposition and in V(D)J recombination might influence the outcomes of these mechanistically similar processes.