Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers

Many genomic processes lead to the formation of underwound (negatively supercoiled) or overwound (positively supercoiled) DNA. These DNA topological changes regulate the interactions of DNA-binding proteins, including transcription factors, architectural proteins and topoisomerases. In order to advance our understanding of the structure and interactions of supercoiled DNA, we recently developed a single-molecule approach called Optical DNA Supercoiling (ODS). This method enables rapid generation of negatively supercoiled DNA (with between <5% and 70% lower helical twist than nonsupercoiled DNA) using a standard dual-trap optical tweezers instrument. ODS is advantageous as it allows for combined force spectroscopy, fluorescence imaging, and spatial control of the supercoiled substrate, which is difficult to achieve with most other approaches. Here, we describe how to generate negatively supercoiled DNA using dual-trap optical tweezers. To this end, we provide detailed instructions on the design and preparation of suitable DNA substrates, as well as a step-by-step guide for how to control and calibrate the supercoiling density produced

Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN

Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches

Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair. While the catalytic cycle of Type 1A topoisomerases has been long-known to involve an enzyme-bridged ssDNA gate that allows strand passage, a deeper mechanistic understanding of these enzymes has only recently begun to emerge. This knowledge has been greatly enhanced through the combination of biochemical studies and increasingly sophisticated single-molecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy and Förster resonance energy transfer. In this review, we discuss how single-molecule assays have advanced our understanding of the gate opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the interplay of Type 1A topoisomerases with partner proteins, such as RecQ-family helicases. We also highlight how these assays have shed new light on the likely functional roles of Type 1A topoisomerases in vivo and discuss recent developments in single-molecule technologies that could be applied to further enhance our understanding of these essential enzymes

Constraints on the origin and evolution of magmas in the Payún Matrú Volcanic Field, Quaternary Andean Back-arc of Western Argentina

The Payún Matrú Volcanic Field (Pleistocene-Holocene) is located in the Andean back-arc of the Southern Volcanic Zone, western Argentina, and is contemporaneous with the Andean volcanic arc at the same latitude. It includes two polygenetic, mostly trachytic volcanoes: Payún Matrú (with a summit caldera 8 km wide) and Payún Liso (a smaller stratovolcano). The volcanic field includes about 200 scoria cones and alkali basaltic and trachybasaltic lava flows, forming two basaltic fields around Payún Matrú. New 40Ar¯39Ar ages extend the activity of Payún Matrú up to 700 ka. The major and trace element and Sr-Nd isotopic compositions of the basaltic lavas and Payún Matrú rocks indicate that the trachytes of Payún Matrú are the result of fractional crystallization of basaltic parent magmas without significant upper crustal contamination, and that the basalts have a geochemical similarity to ocean island basalt (La/Nb=0·8-1·5, La/Ba =0·05-0·08). The Sr-Nd isotopic compositions of the basaltic to trachytic rocks range between 0·703813 and 0·703841 (87Sr/86Sr) and 0·512743 and 0·512834 (143Nd/144Nd). Mass-balance and Rayleigh fractionation models support the proposed origin of the trachytes, and an assimilation-fractional crystallization model indicates a low degree of upper crustal contamination in the youngest trachytes. Magnesium numbers (45-55) and contents of Ni(<20-90 ppm) and Cr (30-180 ppm) in the lavas in the basaltic fields indicate that these are not primary magmas. The data also suggest that the basaltic lavas originated in the asthenospheric mantle, probably within the spinel stability field and beneath an attenuated continental lithosphere in the back-arc area. The lack of a slab-fluid signature in the Payún Matrú Volcanic Field rocks, along with unpublished and published geophysical results (mantle tomography and electrical conductivity anomalies) suggest that magmas were generated by decompression-induced melting of upwelling mantle.Facultad de Ciencias Naturales y MuseoCentro de Investigaciones Geológica

Constraints on the origin and evolution of magmas in the Payún Matrú Volcanic Field, Quaternary Andean Back-arc of Western Argentina

The Payún Matrú Volcanic Field (Pleistocene-Holocene) is located in the Andean back-arc of the Southern Volcanic Zone, western Argentina, and is contemporaneous with the Andean volcanic arc at the same latitude. It includes two polygenetic, mostly trachytic volcanoes: Payún Matrú (with a summit caldera 8 km wide) and Payún Liso (a smaller stratovolcano). The volcanic field includes about 200 scoria cones and alkali basaltic and trachybasaltic lava flows, forming two basaltic fields around Payún Matrú. New 40Ar¯39Ar ages extend the activity of Payún Matrú up to 700 ka. The major and trace element and Sr-Nd isotopic compositions of the basaltic lavas and Payún Matrú rocks indicate that the trachytes of Payún Matrú are the result of fractional crystallization of basaltic parent magmas without significant upper crustal contamination, and that the basalts have a geochemical similarity to ocean island basalt (La/Nb=0·8-1·5, La/Ba =0·05-0·08). The Sr-Nd isotopic compositions of the basaltic to trachytic rocks range between 0·703813 and 0·703841 (87Sr/86Sr) and 0·512743 and 0·512834 (143Nd/144Nd). Mass-balance and Rayleigh fractionation models support the proposed origin of the trachytes, and an assimilation-fractional crystallization model indicates a low degree of upper crustal contamination in the youngest trachytes. Magnesium numbers (45-55) and contents of Ni(<20-90 ppm) and Cr (30-180 ppm) in the lavas in the basaltic fields indicate that these are not primary magmas. The data also suggest that the basaltic lavas originated in the asthenospheric mantle, probably within the spinel stability field and beneath an attenuated continental lithosphere in the back-arc area. The lack of a slab-fluid signature in the Payún Matrú Volcanic Field rocks, along with unpublished and published geophysical results (mantle tomography and electrical conductivity anomalies) suggest that magmas were generated by decompression-induced melting of upwelling mantle.Facultad de Ciencias Naturales y MuseoCentro de Investigaciones Geológica

Seismic and geochemical evidence for large-scale mantle upwelling beneath the eastern Atlantic and western and central Europe

Seismic tomography and the isotope geochemistry of Cenozoic volcanic rocks suggest the existence of a large, sheet-like region of upwelling in the upper mantle which extends from the eastern Atlantic Ocean to central Europe and the western Mediterranean. A belt of extension and rifting in the latter two areas appears to lie above the intersection of the centre of the upwelling region with the base of the lithosphere. Lead, strontium and neodymium isotope data for all three regions converge on a restricted composition, inferred to be that of the upwelling mantle

Сучасний стан і проблеми управління залізничним транспортом України

Проаналізовано стан і тенденції розвитку залізничного транспорту. Розглянуті основні завдання державного регулювання галузі.Проанализировано состояние и тенденции развития железнодорожного транспорта. Рассмотренны основные задания государственного регулирования отрасли.The condition and trends of railway transport has been anilized. The main tasks of state regulation of railway transport has been considereted