BackgroundTransposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; however, current methods are highly sensitive to the amount and quality of input nucleic acid.ResultsWe describe a new library preparation technology (Nextera DNA Flex) that utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA, and enables direct input of blood and saliva using an integrated extraction protocol. We further report results from libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1 ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. Using this bead-linked library preparation method, library yield saturation was observed at an input amount of 100 ng. Preparation of libraries from a range of species with varying GC levels demonstrated uniform coverage of small genomes. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. This library preparation method was also compatible with poor-quality DNA samples, with sequenceable libraries prepared from formalin-fixed paraffin-embedded samples with varying levels of degradation.ConclusionsIn contrast to solution-based library preparation, this bead-based technology produces a normalized, sequencing-ready library for a wide range of DNA input types and amounts, largely obviating the need for DNA quantitation. The robustness of this bead-based library preparation kit and flexibility of input DNA facilitates application across a wide range of fields.

Chromatin “remodeling” is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA–histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be −32kBT for nucleosomes containing unmodified H4 and −27kBT for nucleosomes containing H4-R45H. Thus, the “loosening” or “remodeling” caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HO promoter (in the absence of other remodeling factors), is 5kBT. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.

The conserved MHF1-MHF2 (MHF) complex functions in the activation of the Fanconi anemia (FA) pathway of DNA damage response, in regulating homologous recombination, and in DNA replication fork maintenance. MHF facilitates the processing of multiple types of branched DNAs by the FA DNA translocase FANCM. Here we report the crystal structure of a human MHF-DNA complex that reveals the DNA binding mode of MHF. The structure suggests an MHF preference for branched DNA over double stranded DNA through engaging two duplex arms, which is supported by single molecule studies. Biochemical analyses verify that MHF preferentially engage DNA forks or various four-way junctions independent of the junction-site structure. Genetic experiments provide evidence that the observed DNA-binding interface of MHF is important for cellular resistance to DNA damage. These results provide insights into how the MHF complex recognizes branched DNA and stimulates FANCM activity at such a structure to promote genome maintenance.

Attachments between DNA and a surface or bead are often necessary for single-molecule studies of DNA and DNA-protein interactions. In single-molecule mechanical studies using optical or magnetic tweezers, such attachments must be able to withstand the applied forces. Here we present a new method for covalently attaching DNA to a glass surface, which uses N-hydroxysuccinimide (NHS) modified PEG that is suitable for high-force single-molecule mechanical studies. A glass surface is coated with silane-PEG-NHS and DNA is covalently linked through a reaction between the NHS group and an amine modified nucleotide that has been incorporated into the DNA. After DNA attachment, non-reacted NHS groups are hydrolyzed leaving a PEG-covered surface which has the added benefit of reducing non-specific surface interactions. This method permits specific binding of the DNA to the surface through a covalent bond. At the DNA end not attached to the surface, we attach a streptavidin-coated polystyrene bead and measure force-versus-extension using an optical trap. We show that our method allows a tethered DNA molecule to be pulled through its overstretching transition (>60 pN) multiple times. We anticipate this simple yet powerful method will be useful for many researchers.

We describe a new method for calibrating optical trapping measurements in which tension is applied in the direction of the laser beam to a molecule tethered between a surface and an optically trapped bead. Specifically, we present a generally-applicable procedure for converting from the measured scattering intensity and the measured stage displacement to applied tension and bead-coverslip separation, using measurements of the light intensity scattered from an untethered, trapped bead. Our calibration accounts for a number of effects, including aberrations and the interference of forwardreflected bead-scattered light with the trapping beam. To demonstrate the accuracy of our method, we show measurements of the DNA force-versus-extension relation using a range of laser intensities, and show that these measurements match the expected extensible wormlike-chain (WLC) behavior. Finally, we also demonstrate a force-clamp, in which the tension in a tether is held fixed while the extension varies as a result of molecular events.