The DNA helicase FANCJ (BRIP1) functions in double strand break repair processing, but not crossover formation during prophase I of meiosis in male mice.

Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.

Cathodoluminescence spectroscopy of monolayer hexagonal boron nitride.

Cathodoluminescence (CL) spectroscopy is a suitable technique for studying the luminescent properties of optoelectronic materials because CL has no limitation on the excitable bandgap energy and eliminates ambiguous signals due to simple light scattering and resonant Raman scattering potentially involved in the photoluminescence spectra. However, direct CL measurements of atomically thin two-dimensional materials have been difficult due to the small excitation volume that interacts with high-energy electron beams. Herein, distinct CL signals from a monolayer hexagonal BN (hBN), namely mBN, epitaxial film grown on a graphite substrate are shown by using a CL system capable of large-area and surface-sensitive excitation. Spatially resolved CL spectra at 13 K exhibited a predominant 5.5-eV emission band, which has been ascribed to originate from multilayered aggregates of hBN, markedly at thicker areas formed on the step edges of the substrate. Conversely, a faint peak at 6.04 ± 0.01 eV was routinely observed from atomically flat areas, which is assigned as being due to the recombination of phonon-assisted direct excitons of mBN. The CL results support the transition from indirect bandgap in bulk hBN to direct bandgap in mBN. The results also encourage one to elucidate emission properties of other low-dimensional materials by using the present CL configuration.

PRIMPOL ensures robust handoff between on-the-fly and post-replicative DNA lesion bypass.

The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent 'on-the-fly' lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.

Wafer-Scale Two-Dimensional Semiconductors for Deep UV Sensing.

2D semiconductors (2SEM) can transform many sectors, from information and communication technology to healthcare. To date, top-down approaches to their fabrication, such as exfoliation of bulk crystals by "scotch-tape," are widely used, but have limited prospects for precise engineering of functionalities and scalability. Here, a bottom-up technique based on epitaxy is used to demonstrate high-quality, wafer-scale 2SEM based on the wide band gap gallium selenide (GaSe) compound. GaSe layers of well-defined thickness are developed using a bespoke facility for the epitaxial growth and in situ studies of 2SEM. The dominant centrosymmetry and stacking of the individual van der Waals layers are verified by theory and experiment; their optical anisotropy and resonant absorption in the UV spectrum are exploited for photon sensing in the technological UV-C spectral range, offering a scalable route to deep-UV optoelectronics.

The DNA helicase FANCJ (BRIP1) functions in Double Strand Break repair processing, but not crossover formation during Prophase I of meiosis in male mice.

During meiotic prophase I, recombination between homologous parental chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs), each of which must be repaired with absolute fidelity to ensure genome stability of the germline. One outcome of these DSB events is the formation of Crossovers (COs), the sites of physical DNA exchange between homologs that are critical to ensure the correct segregation of parental chromosomes. However, COs account for only a small (~10%) proportion of all DSB repair events; the remaining 90% are repaired as non-crossovers (NCOs), most by synthesis dependent strand annealing. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers. The number and positioning of COs is exquisitely controlled via mechanisms that remain poorly understood, but which undoubtedly require the coordinated action of multiple repair pathways downstream of the initiating DSB. In a previous report we found evidence suggesting that the DNA helicase and Fanconi Anemia repair protein, FANCJ (BRIP1/BACH1), functions to regulate meiotic recombination in mouse. A gene-trap disruption of Fancj showed an elevated number of MLH1 foci and COs. FANCJ is known to interact with numerous DNA repair proteins in somatic cell repair contexts, including MLH1, BLM, BRCA1, and TOPBP1, and we hypothesized that FANCJ regulates CO formation through a direct interaction with MLH1 to suppress the major CO pathway. To further elucidate the function of FANCJ in meiosis, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, a mutant line lacking the MLH1 interaction site and the N-terminal region of the Helicase domain, and a C-terminal 6xHIS-HA dual-tagged allele of Fancj. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, while Fanconi-like phenotypes are observed within the somatic cell lineages of the full deletion Fancj line, none of the Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I of meiosis. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 during late prophase I. Instead, FANCJ forms discrete foci along the chromosome cores beginning in early meiotic prophase I, occasionally co-localizing with MSH4, and then becomes densely localized on unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Strikingly, this localization strongly overlaps with BRCA1 and TOPBP1. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data suggest a role for FANCJ in early DSB repair events, and possibly in the formation of NCOs, but they rule out a role for FANCJ in MLH1-mediated CO events. Thus, the role of FANCJ in meiotic cells involves different pathways and different interactors to those described in somatic cell lineages.

Optical Monitoring of In Situ Iron Loading into Single, Native Ferritin Proteins.

Ferritin is a protein that stores and releases iron to prevent diseases associated with iron dysregulation in plants, animals, and bacteria. The conversion between iron-loaded holo-ferritin and empty apo-ferritin is an important process for iron regulation. To date, studies of ferritin have used either ensemble measurements to quantify the characteristics of a large number of proteins or single-molecule approaches to interrogate labeled or modified proteins. Here we demonstrate the first real-time study of the dynamics of iron ion loading and biomineralization within a single, unlabeled ferritin protein. Using optical nanotweezers, we trapped single apo- and holo-ferritins indefinitely, distinguished one from the other, and monitored their structural dynamics in real time. The study presented here deepens the understanding of the iron uptake mechanism of ferritin proteins, which may lead to new therapeutics for iron-related diseases.

Creation and resolution of non-B-DNA structural impediments during replication.

During replication, folding of the DNA template into non-B-form secondary structures provides one of the most abundant impediments to the smooth progression of the replisome. The core replisome collaborates with multiple accessory factors to ensure timely and accurate duplication of the genome and epigenome. Here, we discuss the forces that drive non-B structure formation and the evidence that secondary structures are a significant and frequent source of replication stress that must be actively countered. Taking advantage of recent advances in the molecular and structural biology of the yeast and human replisomes, we examine how structures form and how they may be sensed and resolved during replication.

Photo-engineered optoelectronic properties of indium tin oxide via reactive laser annealing.

Transparent conductive oxides are appealing materials for optoelectronic and plasmonic applications as, amongst other advantages, their properties can be modulated by engineering their defects. Optimisation of this adjustment is, however, a complex design problem. This work examined the modification of the carrier transport properties of sputtered tin-doped indium oxide (ITO) via laser annealing in reactive environments. We relate the optical modifications to the structural, compositional, and electronic properties to elucidate the precise mechanisms behind the reactive laser annealing (ReLA) process. For sufficiently high laser fluence, we reveal an ambient-dependent and purely compositional modulation of the carrier concentration of ITO thin films. Hereby, we demonstrate that ReLA utilises the precise energy delivery of photonic processing to enhance the carrier mobility and finely tune the carrier concentration without significantly affecting the crystal structure. Exploitation of this phenomena may enable one to selectively engineer the optoelectronic properties of ITO, promising an alternative to the exploration of new materials for optoelectronic and photonic applications.

Gigavalent Display of Proteins on Monodisperse Polyacrylamide Hydrogels as a Versatile Modular Platform for Functional Assays and Protein Engineering.

The assembly of robust, modular biological components into complex functional systems is central to synthetic biology. Here, we apply modular "plug and play" design principles to a solid-phase protein display system that facilitates protein purification and functional assays. Specifically, we capture proteins on polyacrylamide hydrogel display beads (PHD beads) made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA), that react covalently with SNAP-/Halo-tag fusion proteins, respectively, in a specific, orthogonal, and stable fashion. Anchors, and thus proteins, are distributed throughout the entire bead volume, allowing attachment of ∼109 protein molecules per bead (⌀ 20 µm) -a higher density than achievable with commercial surface-modified beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either noncovalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher, and SnpTag), in mono- and multivalent display formats. Solid-phase protein binding and enzymatic assays are carried out, and incorporating the photocleavable protein PhoCl enables the controlled release of modules via visible-light irradiation for functional assays in solution. We utilize photocleavage for valency engineering of an anti-TRAIL-R1 scFv, enhancing its apoptosis-inducing potency ∼50-fold through pentamerization.

Identifying carbon as the source of visible single-photon emission from hexagonal boron nitride.

Single-photon emitters (SPEs) in hexagonal boron nitride (hBN) have garnered increasing attention over the last few years due to their superior optical properties. However, despite the vast range of experimental results and theoretical calculations, the defect structure responsible for the observed emission has remained elusive. Here, by controlling the incorporation of impurities into hBN via various bottom-up synthesis methods and directly through ion implantation, we provide direct evidence that the visible SPEs are carbon related. Room-temperature optically detected magnetic resonance is demonstrated on ensembles of these defects. We perform ion-implantation experiments and confirm that only carbon implantation creates SPEs in the visible spectral range. Computational analysis of the simplest 12 carbon-containing defect species suggest the negatively charged [Formula: see text] defect as a viable candidate and predict that out-of-plane deformations make the defect environmentally sensitive. Our results resolve a long-standing debate about the origin of single emitters at the visible range in hBN and will be key to the deterministic engineering of these defects for quantum photonic devices.

High-Temperature Molecular Beam Epitaxy of Hexagonal Boron Nitride with High Active Nitrogen Fluxes.

Hexagonal boron nitride (hBN) has attracted a great deal of attention as a key component in van der Waals (vdW) heterostructures, and as a wide band gap material for deep-ultraviolet devices. We have recently demonstrated plasma-assisted molecular beam epitaxy (PA-MBE) of hBN layers on substrates of highly oriented pyrolytic graphite at high substrate temperatures of ~1400 °C. The current paper will present data on the high-temperature PA-MBE growth of hBN layers using a high-efficiency radio-frequency (RF) nitrogen plasma source. Despite more than a three-fold increase in nitrogen flux with this new source, we saw no significant increase in the growth rates of the hBN layers, indicating that the growth rate of hBN layers is controlled by the boron arrival rate. The hBN thickness increases to 90 nm with decrease in the growth temperature to 1080 °C. However, the decrease in the MBE temperature led to a deterioration in the optical properties of the hBN. The optical absorption data indicates that an increase in the active nitrogen flux during the PA-MBE process improves the optical properties of hBN and suppresses defect related optical absorption in the energy range 5.0⁻5.5 eV.

Moiré-Modulated Conductance of Hexagonal Boron Nitride Tunnel Barriers.

Monolayer hexagonal boron nitride (hBN) tunnel barriers investigated using conductive atomic force microscopy reveal moiré patterns in the spatial maps of their tunnel conductance consistent with the formation of a moiré superlattice between the hBN and an underlying highly ordered pyrolytic graphite (HOPG) substrate. This variation is attributed to a periodc modulation of the local density of states and occurs for both exfoliated hBN barriers and epitaxially grown layers. The epitaxial barriers also exhibit enhanced conductance at localized subnanometer regions which are attributed to exposure of the substrate to a nitrogen plasma source during the high temperature growth process. Our results show clearly a spatial periodicity of tunnel current due to the formation of a moiré superlattice and we argue that this can provide a mechanism for elastic scattering of charge carriers for similar interfaces embedded in graphene/hBN resonant tunnel diodes.

Lattice-Matched Epitaxial Graphene Grown on Boron Nitride.

Lattice-matched graphene on hexagonal boron nitride is expected to lead to the formation of a band gap but requires the formation of highly strained material and has not hitherto been realized. We demonstrate that aligned, lattice-matched graphene can be grown by molecular beam epitaxy using substrate temperatures in the range 1600-1710 °C and coexists with a topologically modified moiré pattern with regions of strained graphene which have giant moiré periods up to ∼80 nm. Raman spectra reveal narrow red-shifted peaks due to isotropic strain, while the giant moiré patterns result in complex splitting of Raman peaks due to strain variations across the moiré unit cell. The lattice-matched graphene has a lower conductance than both the Frenkel-Kontorova-type domain walls and also the topological defects where they terminate. We relate these results to theoretical models of band gap formation in graphene/boron nitride heterostructures.

Hexagonal Boron Nitride Tunnel Barriers Grown on Graphite by High Temperature Molecular Beam Epitaxy.

We demonstrate direct epitaxial growth of high-quality hexagonal boron nitride (hBN) layers on graphite using high-temperature plasma-assisted molecular beam epitaxy. Atomic force microscopy reveals mono- and few-layer island growth, while conducting atomic force microscopy shows that the grown hBN has a resistance which increases exponentially with the number of layers, and has electrical properties comparable to exfoliated hBN. X-ray photoelectron spectroscopy, Raman microscopy and spectroscopic ellipsometry measurements on hBN confirm the formation of sp2-bonded hBN and a band gap of 5.9 ± 0.1 eV with no chemical intermixing with graphite. We also observe hexagonal moiré patterns with a period of 15 nm, consistent with the alignment of the hBN lattice and the graphite substrate.

Strain-Engineered Graphene Grown on Hexagonal Boron Nitride by Molecular Beam Epitaxy.

Graphene grown by high temperature molecular beam epitaxy on hexagonal boron nitride (hBN) forms continuous domains with dimensions of order 20 µm, and exhibits moiré patterns with large periodicities, up to ~30 nm, indicating that the layers are highly strained. Topological defects in the moiré patterns are observed and attributed to the relaxation of graphene islands which nucleate at different sites and subsequently coalesce. In addition, cracks are formed leading to strain relaxation, highly anisotropic strain fields, and abrupt boundaries between regions with different moiré periods. These cracks can also be formed by modification of the layers with a local probe resulting in the contraction and physical displacement of graphene layers. The Raman spectra of regions with a large moiré period reveal split and shifted G and 2D peaks confirming the presence of strain. Our work demonstrates a new approach to the growth of epitaxial graphene and a means of generating and modifying strain in graphene.

High broad-band photoresponsivity of mechanically formed InSe-graphene van der Waals heterostructures.

High broad-band photoresponsivity of mechanically formed InSe-graphene van der Waals heterostructures is achieved by exploiting the broad-band transparency of graphene, the direct bandgap of InSe, and the favorable band line up of InSe with graphene. The photoresponsivity exceeds that for other van der Waals heterostructures and the spectral response extends from the near-infrared to the visible spectrum.

Growth and tumor suppressor NORE1A is a regulatory node between Ras signaling and microtubule nucleation.

NORE1A is a Ras-binding protein that belongs to a group of tumor suppressors known as the Ras association domain family. Their growth- and tumor-suppressive function is assumed to be dependent on association with the microtubule cytoskeleton. However, a detailed understanding of this interplay is still missing. Here, we show that NORE1A directly interacts with tubulin and is capable of nucleating microtubules. Strikingly, the ability to stimulate nucleation is regulated in a dual specific way either via phosphorylation of NORE1A within the Ras-binding domain by Aurora A kinase or via binding to activated Ras. We also demonstrate that NORE1A mediates a negative effect of activated Ras on microtubule nucleation. On the basis of our results, we propose a novel regulatory network composed of the tumor suppressor NORE1A, the mitotic kinase Aurora A, the small GTPase Ras, and the microtubule cytoskeleton.

Polarization effects in optically bound particle arrays.

Sub-micron polystyrene spheres spontaneously assemble into twodimensional arrays in the evanescent field of counterpropagating laser beams at the silica-water interface. The symmetry and dynamics of these arrays depends on the particle size and the polarization of the two laser beams. Here we describe the polarization effects for particles with diameters of 390-520 nm, which are small enough to form regular 2-D arrays yet large enough to be readily observed with an optical microscope. We report the observation of rectangular arrays, three different types of hexagonal arrays and a defective array in which every third row is missing. The structure of the arrays is determined by both optical trapping and optical binding. Optical binding can overwhelm optical trapping and give rise to an array that is incommensurate with the interference fringes formed by two laser beams of the same polarization.