RESUMO
The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.
Assuntos
DNA Cruciforme , DNA , DNA/química , Replicação do DNA , Eletroforese em Gel de PoliacrilamidaRESUMO
Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/enzimologia , DNA de Helmintos/metabolismo , RecQ Helicases/fisiologia , Recombinação Genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Quebras de DNA de Cadeia Dupla , DNA Complementar/genética , DNA Cruciforme , DNA de Helmintos/genética , Humanos , Hidrólise , Meiose , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fase S , Especificidade por SubstratoRESUMO
A plausible consequence of the rugged folding energy landscapes inherent to biomolecules is that there may be more than one functionally competent folded state. Indeed, molecule-to-molecule variations in the folding dynamics of enzymes and ribozymes have recently been identified in single-molecule experiments, but without systematic quantification or an understanding of their structural origin. Here, using concepts from glass physics and complementary clustering analysis, we provide a quantitative method to analyse single-molecule fluorescence resonance energy transfer (smFRET) data, thereby probing the isomerization dynamics of Holliday junctions, which display such heterogeneous dynamics over a long observation time (T(obs) ≈ 40 s). We show that the ergodicity of Holliday junction dynamics is effectively broken and that their conformational space is partitioned into a folding network of kinetically disconnected clusters. Theory suggests that the persistent heterogeneity of Holliday junction dynamics is a consequence of internal multiloops with varying sizes and flexibilities frozen by Mg(2+) ions. An annealing experiment using Mg(2+) pulses lends support to this idea by explicitly showing that interconversions between trajectories with different patterns can be induced.
Assuntos
DNA Cruciforme/química , Transferência Ressonante de Energia de Fluorescência , Algoritmos , Sequência de Bases , DNA Cruciforme/genética , Isomerismo , Magnésio/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , TermodinâmicaRESUMO
Helicases are ubiquitous enzymes that disrupt complementary strands of duplex nucleic acid in a reaction dependent on nucleoside-5'-triphosphate hydrolysis. Helicases are implicated in the metabolism of DNA structures that are generated during replication, recombination, and DNA repair. Furthermore, an increasing number of helicases have been linked to genomic instability and human disease. With the growing interest in helicase mechanism and function, we have set out to describe some basic protocols for biochemical characterization of DNA helicases. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding activity of DNA helicases are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of a given helicase, which would serve as a platform for further investigation of its molecular and cellular functions.