RESUMEN
DNA transactions in eukaryotes require that proteins gain access to target sequences packaged in chromatin. Further, interactions between distinct nucleoprotein complexes are often required to generate higher-order structures. Here, we employed two prokaryotic site-specific recombination systems to investigate how chromatin packaging affects the assembly of nucleoprotein structures of different complexities at more than 30 genomic loci. The dynamic nature of chromatin permitted protein-DNA and DNA-DNA interactions for sites of at least 34 bp in length. However, the assembly of higher-order nucleoprotein structures on targets spanning 114 bp was impaired. This impediment was maintained over at least 72 h and was not affected by the transcriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases. Our findings suggest that nucleosomal linker-sized DNA segments become accessible within hours for protein binding due to the dynamic nature of chromatin. Longer segments, however, appear refractory for complete occupancy by sequence-specific DNA-binding proteins. The results thus also provide an explanation why simple recombination systems such as Cre and Flp are proficient in eukaryotic chromatin.
Asunto(s)
Cromatina/genética , Nucleoproteínas/genética , Recombinación Genética , Animales , Sitios de Unión , Células CHO , Cromatina/química , Cromatina/metabolismo , Cricetinae , Inhibidores de Histona Desacetilasas , Integrasas , Sustancias Macromoleculares , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Recombinasas , Inhibidores de Topoisomerasa , Transposasas , Proteínas ViralesRESUMEN
We have transferred the prokaryotic gammadelta resolvase system to mammalian cells and present a comparative analysis of recombination by wild-type and two mutant resolvases (E124Q and E102Y/E124Q). Transient co-transfection assays using beta-galactosidase as reporter for recombination reveal that episomal DNA does not contain a significant level of unconstrained negative supercoiling, since only mutant resolvases are recombination-proficient. We also show that the efficiency of recombination by the resolvase double mutant is comparable to that observed with Cre, which indicates that resolvase can be used as a new tool for controlled manipulations of episomal DNAs.
Asunto(s)
Escherichia coli/enzimología , Mutación/genética , Conformación de Ácido Nucleico , Plásmidos/química , Recombinación Genética/genética , Transposasas/metabolismo , Proteínas Virales , Animales , Sitios de Ligazón Microbiológica/genética , Células CHO , Catálisis , Cricetinae , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Escherichia coli/genética , Genes Reporteros/genética , Integrasas/genética , Integrasas/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Recombinasas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transfección , Transposasas/genéticaRESUMEN
Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-specific recombinases, which includes Cre and FLP. The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination. Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells. Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wild-type Int, however, is not detectable by this method. The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells.
Asunto(s)
Bacteriófago lambda/enzimología , Integrasas/metabolismo , Mutación/genética , Recombinación Genética/genética , Sitios de Ligazón Microbiológica/genética , Bacteriófago lambda/genética , Southern Blotting , Catálisis , Línea Celular , ADN Superhelicoidal/genética , Genoma Humano , Células HeLa , Humanos , Integrasas/genética , Transfección , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
DNA resolvases and invertases are closely related, yet catalyze recombination within two distinct nucleoprotein structures termed synaptosomes and invertasomes, respectively. Different protein-protein and protein-DNA interactions guide the assembly of each type of recombinogenic complex, as well as the subsequent activation of DNA strand exchange. Here we show that invertase Gin catalyzes factor for inversion stimulation dependent inversion on isolated copies of sites I from ISXc5 res, which is typically utilized by the corresponding resolvase. The concomitant binding of Gin to sites I and III in res, however, inhibits recombination. A chimeric recombinase, composed of the catalytic domain of Gin and the DNA-binding domain of ISXc5 resolvase, recombines two res with high efficiency. Gin must therefore contain residues proficient for both synaptosome formation and activation of strand exchange. Surprisingly, this chimera is unable to assemble a productive invertasome; a result which implies a role for the C-terminal domain in invertasome formation that goes beyond DNA binding.
