RESUMO
Repetitive extragenic palindrome (REP)-associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5'-G and 3'-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Sequências Repetidas Invertidas/genética , Transposases/química , Transposases/metabolismoRESUMO
The process of DNA transposition involves the binding, cleavage, and recombination of specific DNA segments (transposable elements, TE) and is catalyzed by special enzymes encoded by the TE transposases. REP-associated tyrosine transposases (RAYTs) are a class of Y1 nucleases related to the IS200/IS605 transposases associated with a bacterial TE known as repetitive extragenic palindrome elements (REPs). Although RAYT has been subject of numerous studies, where DNA binding and cleavage by RAYT have been confirmed for Escherichia coli, the molecular mechanism of DNA insertion has not been fully understood. In this work, it is demonstrated that surface plasmon resonance (SPR) biosensor technology combined with a system of DNA hairpin probes (mimicking the natural REP sequence) and short oligonucleotides (ONs) can provide a rapid and real-time platform for monitoring and quantification of RAYT activity. We utilized RAYT from E. coli (strain MG1655) as a model system, where we evaluated its activity towards both a natural REP sequence as well as REP sequences having modifications targeting specific features of the DNA crucial for the DNA binding and cleavage. The characteristics of the RAYT-DNA interaction obtained by means of the SPR approach were compared with the results of SDS-PAGE analysis.
Assuntos
Técnicas Biossensoriais/instrumentação , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Ressonância de Plasmônio de Superfície/instrumentação , Transposases/metabolismo , Técnicas Biossensoriais/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Ressonância de Plasmônio de Superfície/métodos , Transposases/química , Transposases/genéticaRESUMO
DNA is a structurally plastic molecule, and its biological function is enabled by adaptation to its binding partners. To identify the DNA structural polymorphisms that are possible in such adaptations, the dinucleotide structures of 60â 000 DNA steps from sequentially nonredundant crystal structures were classified and an automated protocol assigning 44 distinct structural (conformational) classes called NtC (for Nucleotide Conformers) was developed. To further facilitate understanding of the DNA structure, the NtC were assembled into the DNA structural alphabet CANA (Conformational Alphabet of Nucleic Acids) and the projection of CANA onto the graphical representation of the molecular structure was proposed. The NtC classification was used to define a validation score called confal, which quantifies the conformity between an analyzed structure and the geometries of NtC. NtC and CANA assignment were applied to analyze the structural properties of typical DNA structures such as Dickerson-Drew dodecamers, guanine quadruplexes and structural models based on fibre diffraction. NtC, CANA and confal assignment, which is accessible at the website https://dnatco.org, allows the quantitative assessment and validation of DNA structures and their subsequent analysis by means of pseudo-sequence alignment. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Acta_Cryst_D:2.
Assuntos
DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Gráficos por Computador , Simulação de Dinâmica MolecularRESUMO
Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8â Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π-cation motif of stacked residues KWRWRH, a NAG-W-NAG sandwich (where NAG stands for N-acetyl-D-glucosamine) and finally a helix formed by residues 78-85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2.