RESUMO
The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model's ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.
Assuntos
Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Lisogenia/genética , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação Viral/genética , Sítios de Ligação , Simulação por Computador , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Cruciforme/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Transferência Ressonante de Energia de Fluorescência , Integrases/química , Integrases/genética , Integrases/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Nucleoproteínas/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Recombinação Genética , Proteínas Virais/químicaRESUMO
The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination.
Assuntos
Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Integrases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sítios de Ligação , Reagentes de Ligações Cruzadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Cruciforme/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Integrases/química , Integrases/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Recombinação Genética , Proteínas Virais/química , Ativação Viral/genética , Integração Viral/genéticaRESUMO
Prion variants are self-perpetuating conformers of a single protein that assemble into amyloid fibers and confer unique phenotypic states. Multiple prion variants can arise, particularly in response to changing environments, and interact within an organism. These interactions are often competitive, with one variant establishing phenotypic dominance over the others. This dominance has been linked to the competition for non-prion state protein, which must be converted to the prion state via a nucleated polymerization mechanism. However, the intrinsic rates of conversion, determined by the conformation of the variant, cannot explain prion variant dominance, suggesting a more complex interaction. Using the yeast prion system [PSI+ ], we have determined the mechanism of dominance of the [PSI+ ]Strong variant over the [PSI+ ]Weak variant in vivo. When mixed by mating, phenotypic dominance is established in zygotes, but the two variants persist and co-exist in the lineage descended from this cell. [PSI+ ]Strong propagons, the heritable unit, are amplified at the expense of [PSI+ ]Weak propagons, through the efficient conversion of soluble Sup35 protein, as revealed by fluorescence photobleaching experiments employing variant-specific mutants of Sup35. This competition, however, is highly sensitive to the fragmentation of [PSI+ ]Strong amyloid fibers, with even transient inhibition of the fragmentation catalyst Hsp104 promoting amplification of [PSI+ ]Weak propagons. Reducing the number of [PSI+ ]Strong propagons prior to mating, similarly promotes [PSI+ ]Weak amplification and conversion of soluble Sup35, indicating that template number and conversion efficiency combine to determine dominance. Thus, prion variant dominance is not an absolute hierarchy but rather an outcome arising from the dynamic interplay between unique protein conformations and their interactions with distinct cellular proteostatic niches.
RESUMO
Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Förster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the λ integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA.
Assuntos
DNA Super-Helicoidal/química , Recombinação Genética , Sítios de Ligação Microbiológicos , DNA Cruciforme/metabolismo , DNA Super-Helicoidal/metabolismo , Fluoresceína , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Técnicas Genéticas , Origem de Replicação , RodaminasRESUMO
The transparent nematode Caenorhabditis elegans can sense UV and blue-violet light to alter behavior. Because high-dose UV and blue-violet light are not a common feature outside of the laboratory setting, we asked what role, if any, could low-intensity visible light play in C. elegans physiology and longevity. Here, we show that C. elegans lifespan is inversely correlated to the time worms were exposed to visible light. While circadian control, lite-1 and tax-2 do not contribute to the lifespan reduction, we demonstrate that visible light creates photooxidative stress along with a general unfolded-protein response that decreases the lifespan. Finally, we find that long-lived mutants are more resistant to light stress, as well as wild-type worms supplemented pharmacologically with antioxidants. This study reveals that transparent nematodes are sensitive to visible light radiation and highlights the need to standardize methods for controlling the unrecognized biased effect of light during lifespan studies in laboratory conditions.
Assuntos
Caenorhabditis elegans/efeitos da radiação , Luz/efeitos adversos , Longevidade/efeitos da radiação , Estresse Oxidativo , Acetilcisteína , Animais , Antioxidantes , Ácido Ascórbico , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Fotoperíodo , Resposta a Proteínas não DobradasRESUMO
The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.
Assuntos
Bacteriófago lambda/genética , DNA Bacteriano/química , DNA Cruciforme/química , DNA Viral/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Recombinação Genética , Microscopia Crioeletrônica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Imageamento Tridimensional , Modelos MolecularesRESUMO
Autophagy-dependent longevity models in C. elegans display altered lipid storage profiles, but the contribution of lipid distribution to life-span extension is not fully understood. Here we report that lipoprotein production, autophagy and lysosomal lipolysis are linked to modulate life span in a conserved fashion. We find that overexpression of the yolk lipoprotein VIT/vitellogenin reduces the life span of long-lived animals by impairing the induction of autophagy-related and lysosomal genes necessary for longevity. Accordingly, reducing vitellogenesis increases life span via induction of autophagy and lysosomal lipolysis. Life-span extension due to reduced vitellogenesis or enhanced lysosomal lipolysis requires nuclear hormone receptors (NHRs) NHR-49 and NHR-80, highlighting novel roles for these NHRs in lysosomal lipid signaling. In dietary-restricted worms and mice, expression of VIT and hepatic APOB (apolipoprotein B), respectively, are significantly reduced, suggesting a conserved longevity mechanism. Altogether, our study demonstrates that lipoprotein biogenesis is an important mechanism that modulates aging by impairing autophagy and lysosomal lipolysis.
Assuntos
Autofagia , Caenorhabditis elegans/fisiologia , Lipoproteínas/biossíntese , Longevidade/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Restrição Calórica , Regulação da Expressão Gênica , Inativação Gênica , Mucosa Intestinal/metabolismo , Lipase/metabolismo , Lipólise , Lisossomos/metabolismo , Transcrição Gênica , Vitelogênese/genética , Vitelogeninas/metabolismoRESUMO
Multivalent glycoconjugates were prepared using generation-4 PAMAM dendrimers, and their interaction with Langmuir monolayers containing GM3 was investigated. Excessive carbohydrate valency adversely affects the carbohydrate-carbohydrate interaction. The GM3 monolayer selectively interacts with lactose-functionalized dendrimers in the presence of calcium ions.