Chk2 homologue Mek1 limits Exo1-dependent DNA end resection during meiotic recombination in S. cerevisiae.

The conserved Rad2/XPG family 5'-3' exonuclease, Exonuclease 1 (Exo1), plays many roles in DNA metabolism including during resolution of DNA double strand breaks (DSBs) via homologous recombination. Prior studies provided evidence that the end-resection activity of Exo1 is downregulated in yeast and mammals by Cdk1/2 family cyclin-dependent and checkpoint kinases, including budding yeast kinase Rad53 which functions in mitotic cells. Here we provide evidence that the master meiotic kinase Mek1, a paralogue of Rad53, limits 5'-3' single strand resection at the sites of programmed meiotic DNA breaks. Mutational analysis suggests that the mechanism of Exo1 suppression by Mek1 differs from that of Rad53. Article Summary: Meiotic recombination involves formation of programmed DNA double strand breaks followed by 5' to 3' single strand specific resection by nucleases including Exo1. We find that the activity of budding yeast Exo1 is downregulated during meiotic recombination by the master meiotic kinase Mek1. The mechanism of downregulation of Exo1 by Mek1 in meiosis does not depend on the same phospho-sites as those used by the mitotic kinase Rad53, a relative of Mek1 that downregulates Exo1 in mitosis.

How strand exchange protein function benefits from ATP hydrolysis.

Members of the RecA family of strand exchange proteins carry out the central reaction in homologous recombination. These proteins are DNA-dependent ATPases, although their ATPase activity is not required for the key functions of homology search and strand exchange. We review the literature on the role of the intrinsic ATPase activity of strand exchange proteins. We also discuss the role of ATP-hydrolysis-dependent motor proteins that serve as strand exchange accessory factors, with an emphasis on the eukaryotic Rad54 family of double strand DNA-specific translocases. The energy from ATP allows recombination events to progress from the strand exchange stage to subsequent stages. ATP hydrolysis also functions to corrects DNA binding errors, including particularly detrimental binding to double strand DNA.

A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability.

During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes ("homologs"), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1's activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.

Carrier-resolved photo-Hall effect.

The fundamental parameters of majority and minority charge carriers-including their type, density and mobility-govern the performance of semiconductor devices yet can be difficult to measure. Although the Hall measurement technique is currently the standard for extracting the properties of majority carriers, those of minority carriers have typically only been accessible through the application of separate techniques. Here we demonstrate an extension to the classic Hall measurement-a carrier-resolved photo-Hall technique-that enables us to simultaneously obtain the mobility and concentration of both majority and minority carriers, as well as the recombination lifetime, diffusion length and recombination coefficient. This is enabled by advances in a.c.-field Hall measurement using a rotating parallel dipole line system and an equation, ΔµH = d(σ2H)/dσ, which relates the hole-electron Hall mobility difference (ΔµH), the conductivity (σ) and the Hall coefficient (H). We apply this technique to various solar absorbers-including high-performance lead-iodide-based perovskites-and demonstrate simultaneous access to majority and minority carrier parameters and map the results against varying light intensities. This information, which is buried within the photo-Hall measurement1,2, had remained inaccessible since the original discovery of the Hall effect in 18793. The simultaneous measurement of majority and minority carriers should have broad applications, including in photovoltaics and other optoelectronic devices.

Distinct Functions in Regulation of Meiotic Crossovers for DNA Damage Response Clamp Loader Rad24(Rad17) and Mec1(ATR) Kinase.

The number and distribution of meiotic crossovers (COs) are highly regulated, reflecting the requirement for COs during the first round of meiotic chromosome segregation. CO control includes CO assurance and CO interference, which promote at least one CO per chromosome bivalent and evenly-spaced COs, respectively. Previous studies revealed a role for the DNA damage response (DDR) clamp and the clamp loader in CO formation by promoting interfering COs and interhomolog recombination, and also by suppressing ectopic recombination. In this study, we use classical tetrad analysis of Saccharomyces cerevisiae to show that a mutant defective in RAD24, which encodes the DDR clamp loader (RAD17 in other organisms), displayed reduced CO frequencies on two shorter chromosomes (III and V), but not on a long chromosome (chromosome VII). The residual COs in the rad24 mutant do not show interference. In contrast to rad24, mutants defective in the ATR kinase homolog Mec1, including a mec1 null and a mec1 kinase-dead mutant, show slight or few defects in CO frequency. On the other hand, mec1 COs show defects in interference, similar to the rad24 mutant. Our results support a model in which the DDR clamp and clamp-loader proteins promote interfering COs by recruiting pro-CO Zip, Mer, and Msh proteins to recombination sites, while the Mec1 kinase regulates CO distribution by a distinct mechanism. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

Non-enzymatic roles of human RAD51 at stalled replication forks.

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not D-loop activity, to reveal mechanistic aspects of RAD51's roles in the response to replication stress. Here, we find that cells lacking RAD51's enzymatic activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly, we find that RAD51's strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork regression, supporting a model in which fork regression depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

Improving Carrier-Transport Properties of CZTS by Mg Incorporation with Spray Pyrolysis.

High nonradiative recombination, low diffusion length and band tailing are often associated with a large open circuit voltage deficit, which results in low efficiency of Cu2ZnSnS4 (CZTS) solar cells. Recently, cation substitution in CZTS has gained interest as a plausible solution to suppress these issues. However, the common substitutes, Ag and Cd, are not ideal due to their scarcity and toxicity. Other transition-metal candidates (e.g., Mn, Fe, Co, or Ni) are multivalent, which may form harmful deep-level defects. Magnesium, as one of the viable substitutes, does not have these issues, as it is very stable in +2 oxidation state, abundant, and nontoxic. In this study, we investigate the effect of Mg incorporation in sulfur-based Cu2ZnSnS4 to form Cu2MgxZn1-xSnS4 by varying x from 0.0 to 1.0. These films were fabricated by chemical spray pyrolysis and the subsequent sulfurization process. At a high Mg content, it is found that Mg does not replace Zn to form a quaternary compound, which leads to the appearance of the secondary phases in the sample. However, a low Mg content (Cu2Mg0.05Zn0.95SnS4) improves the power conversion efficiency from 5.10% (CZTS) to 6.73%. The improvement is correlated to the better carrier-transport properties, as shown by a lesser amount of the ZnS secondary phase, higher carrier mobility, and shallower acceptor defects level. In addition, the Cu2Mg0.05Zn0.95SnS4 device also shows better charge-collection property based on the higher fill factor and quantum efficiency despite having lower depletion width. Therefore, we believe that the addition of a small amount of Mg is another viable route to improve the performance of the CZTS solar cell.

RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination.

Dmc1 catalyzes homology search and strand exchange during meiotic recombination in budding yeast and many other organisms including humans. Here we reconstitute Dmc1 recombination in vitro using six purified proteins from budding yeast including Dmc1 and its accessory proteins RPA, Rad51, Rdh54/Tid1, Mei5-Sae3 and Hop2-Mnd1 to promote D-loop formation between ssDNA and dsDNA substrates. Each accessory protein contributed to Dmc1's activity, with the combination of all six proteins yielding optimal activity. The ssDNA binding protein RPA plays multiple roles in stimulating Dmc1's activity including by overcoming inhibitory effects of ssDNA secondary structure on D-loop reactions, and by elongating D-loops. In addition, we demonstrate that RPA limits inhibitory interactions of Hop2-Mnd1 and Rdh54/Tid1 that otherwise occur during assembly of Dmc1-ssDNA nucleoprotein filaments. Finally, we report interactions between the proteins employed in the biochemical reconstitution including a direct interaction between Rad51 and Dmc1 that is enhanced by Mei5-Sae3.

Intrinsically ultrastrong plasmon-exciton interactions in crystallized films of carbon nanotubes.

In cavity quantum electrodynamics, optical emitters that are strongly coupled to cavities give rise to polaritons with characteristics of both the emitters and the cavity excitations. We show that carbon nanotubes can be crystallized into chip-scale, two-dimensionally ordered films and that this material enables intrinsically ultrastrong emitter-cavity interactions: Rather than interacting with external cavities, nanotube excitons couple to the near-infrared plasmon resonances of the nanotubes themselves. Our polycrystalline nanotube films have a hexagonal crystal structure, ∼25-nm domains, and a 1.74-nm lattice constant. With this extremely high nanotube density and nearly ideal plasmon-exciton spatial overlap, plasmon-exciton coupling strengths reach 0.5 eV, which is 75% of the bare exciton energy and a near record for room-temperature ultrastrong coupling. Crystallized nanotube films represent a milestone in nanomaterials assembly and provide a compelling foundation for high-ampacity conductors, low-power optical switches, and tunable optical antennas.

The ATPase activity of E. coli RecA prevents accumulation of toxic complexes formed by erroneous binding to undamaged double stranded DNA.

The Escherichia coli RecA protein catalyzes the central step of homologous recombination using its homology search and strand exchange activity. RecA is a DNA-dependent ATPase, but its homology search and strand exchange activities are largely independent of its ATPase activity. ATP hydrolysis converts a high affinity DNA binding form, RecA-ATP, to a low affinity form RecA-ADP, thereby supporting an ATP hydrolysis-dependent dynamic cycle of DNA binding and dissociation. We provide evidence for a novel function of RecA's dynamic behavior; RecA's ATPase activity prevents accumulation of toxic complexes caused by direct binding of RecA to undamaged regions of dsDNA. We show that a mutant form of RecA, RecA-K250N, previously shown to be toxic to E. coli, is a loss-of-function ATPase-defective mutant. We use a new method for detecting RecA complexes involving nucleoid surface spreading and immunostaining. The method allows detection of damage-induced RecA foci; STED microscopy revealed these to typically be between 50 and 200 nm in length. RecA-K250N, and other toxic variants of RecA, form spontaneous DNA-bound complexes that are independent of replication and of accessory proteins required to load RecA onto tracts of ssDNA in vivo, supporting the hypothesis that RecA's expenditure of ATP serves an error correction function.

Purification of Saccharomyces cerevisiae Homologous Recombination Proteins Dmc1 and Rdh54/Tid1 and a Fluorescent D-Loop Assay.

Budding yeast Dmc1 is a member of the RecA family of strand exchange proteins essential for homologous recombination (HR) during meiosis. Dmc1 mediates the steps of homology search and DNA strand exchange reactions that are central to HR. To achieve optimum activity, Dmc1 requires a number of accessory factors. Although methods for purification of Dmc1 and many of its associated factors have been described (Binz, Dickson, Haring, & Wold, 2006; Busygina et al., 2013; Chan, Brown, Qin, Handa, & Bishop, 2014; Chi et al., 2006; Cloud, Chan, Grubb, Budke, & Bishop, 2012; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Van Komen, Macris, Sehorn, & Sung, 2006), Dmc1 has been particularly difficult to purify because of its tendency to aggregate. Here, we provide an alternative and simple high-yield purification method for recombinant Dmc1 that is active and responsive to stimulation by accessory factors. The same method may be used for purification of recombinant Rdh54 (a.k.a. Tid1) and other HR proteins with minor adjustments. We also describe an economical and sensitive D-loop assay for strand exchange proteins that uses fluorescent dye-tagged, rather than radioactive, ssDNA substrates.

Ultrathin high band gap solar cells with improved efficiencies from the world's oldest photovoltaic material.

Selenium was used in the first solid state solar cell in 1883 and gave early insights into the photoelectric effect that inspired Einstein's Nobel Prize work; however, the latest efficiency milestone of 5.0% was more than 30 years ago. The recent surge of interest towards high-band gap absorbers for tandem applications led us to reconsider this attractive 1.95 eV material. Here, we show completely redesigned selenium devices with improved back and front interfaces optimized through combinatorial studies and demonstrate record open-circuit voltage (V OC) of 970 mV and efficiency of 6.5% under 1 Sun. In addition, Se devices are air-stable, non-toxic, and extremely simple to fabricate. The absorber layer is only 100 nm thick, and can be processed at 200 ˚C, allowing temperature compatibility with most bottom substrates or sub-cells. We analyze device limitations and find significant potential for further improvement making selenium an attractive high-band-gap absorber for multi-junction device applications.Wide band gap semiconductors are important for the development of tandem photovoltaics. By introducing buffer layers at the front and rear side of solar cells based on selenium; Todorov et al., reduce interface recombination losses to achieve photoconversion efficiencies of 6.5%.

Nanoscale Characterization of Back Surfaces and Interfaces in Thin-Film Kesterite Solar Cells.

Combinations of sub 1 µm absorber films with high-work-function back surface contact layers are expected to induce large enough internal fields to overcome adverse effects of bulk defects on thin-film photovoltaic performance, particularly in earth-abundant kesterites. However, there are numerous experimental challenges involving back surface engineering, which includes exfoliation, thinning, and contact layer optimization. In the present study, a unique combination of nanocharacterization tools, including nano-Auger, Kelvin probe force microscopy (KPFM), and cryogenic focused ion beam measurements, are employed to gauge the possibility of surface potential modification in the absorber back surface via direct deposition of high-work-function metal oxides on exfoliated surfaces. Nano-Auger measurements showed large compositional nonuniformities on the exfoliated surfaces, which can be minimized by a brief bromine-methanol etching step. Cross-sectional nano-Auger and KPFM measurements on Au/MoO3/Cu2ZnSn(S,Se)4 (CZTSSe) showed an upward band bending as large as 400 meV within the CZTSSe layer, consistent with the high work function of MoO3, despite Au incorporation into the oxide layer. Density functional theory simulations of the atomic structure for bulk amorphous MoO3 demonstrated the presence of large voids within MoO3 enabling Au in-diffusion. With a less diffusive metal electrode such as Pt or Pd, upward band bending beyond this level is expected to be achieved.

Surface Spreading and Immunostaining of Yeast Chromosomes.

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2.

In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5' to 3' end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5' to 3' resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA.

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1(ATR)/Tel1(ATM)-dependent DNA damage response or caffeine's inhibition of 5' to 3' resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.

DNA damage response clamp 9-1-1 promotes assembly of ZMM proteins for formation of crossovers and synaptonemal complex.

Formation of crossovers between homologous chromosomes during meiosis is positively regulated by the ZMM proteins (also known as SIC proteins). DNA damage checkpoint proteins also promote efficient formation of interhomolog crossovers. Here, we examined, in budding yeast, the meiotic role of the heterotrimeric DNA damage response clamp composed of Rad17, Ddc1 and Mec3 (known as '9-1-1' in other organisms) and a component of the clamp loader, Rad24 (known as Rad17 in other organisms). Cytological analysis indicated that the 9-1-1 clamp and its loader are not required for the chromosomal loading of RecA homologs Rad51 or Dmc1, but are necessary for the efficient loading of ZMM proteins. Interestingly, the loading of ZMM proteins onto meiotic chromosomes was independent of the checkpoint kinase Mec1 (the homolog of ATR) as well as Rad51. Furthermore, the ZMM member Zip3 (also known as Cst9) bound to the 9-1-1 complex in a cell-free system. These data suggest that, in addition to promoting interhomolog bias mediated by Rad51-Dmc1, the 9-1-1 clamp promotes crossover formation through a specific role in the assembly of ZMM proteins. Thus, the 9-1-1 complex functions to promote two crucial meiotic recombination processes, the regulation of interhomolog recombination and crossover formation mediated by ZMM.

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.

Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome.