Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology.

The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51-DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4 nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10 nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3'end of ssDNA or from a region adjacent to the ss-ds junction. RAD51 filaments covering joint ss-dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange.

Effect of the BRCA2 CTRD domain on RAD51 filaments analyzed by an ensemble of single molecule techniques.

Homologous recombination is essential for the preservation of genome stability, thereby preventing cancer. The recombination protein RAD51 drives DNA strand exchange, which requires the assembly, rearrangement and disassembly of a RAD51 filament on DNA, coupled to ATP binding and hydrolysis. This process is facilitated and controlled by recombination mediators and accessory factors. Here, we have employed a range of single molecule techniques to determine the influence of the C-terminal RAD51 interaction domain (CTRD) of the breast cancer tumor suppressor BRCA2 on intrinsic aspects of RAD51-DNA interactions. We show that at high concentration the CTRD entangles RAD51 filaments and reduces RAD51 filament formation in a concentration dependent manner. It does not affect the rate of filament disassembly measured as the loss of fluorescent signal due to intrinsic RAD51 protein dissociation from double-stranded DNA (dsDNA). We conclude that, outside the context of the full-length protein, the CTRD does not reduce RAD51 dissociation kinetics, but instead hinders filament formation on dsDNA. The CTRDs mode of action is most likely sequestration of multiple RAD51 molecules thereby rendering them inactive for filament formation on dsDNA.

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies.

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).

Fuel ethanol from cellulosic biomass.

Ethanol produced from cellulosic biomass is examined as a large-scale transportation fuel. Desirable features include ethanol's fuel properties as well as benefits with respect to urban air quality, global climate change, balance of trade, and energy security. Energy balance, feedstock supply, and environmental impact considerations are not seen as significant barriers to the widespread use of fuel ethanol derived from cellulosic biomass. Conversion economics is the key obstacle to be overcome. In light of past progress and future prospects for research-driven improvements, a cost-competitive process appears possible in a decade.

Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein.

Nitrogen regulatory protein C (NtrC) contacts a bacterial RNA polymerase from distant enhancers by means of DNA loops and activates transcription by allowing polymerase to gain access to the template DNA strand. It was shown that NtrC from Salmonella typhimurium must build large oligomers to activate transcription. In contrast to eukaryotic enhancer-binding proteins, most of which must bind directly to DNA, some NtrC dimers were bound solely by protein-protein interactions. NtrC oligomers were visualized with scanning force microscopy. Evidence of their functional importance was provided by showing that some inactive non-DNA-binding and DNA-binding mutant forms of NtrC can cooperate to activate transcription.

DNA replication. A familiar ring to DNA polymerase processivity.

Structural similarity reveals that prokaryotic and eukaryotic DNA polymerases share a mechanism for processivity--but the conservation of additional chromosomal replication mechanisms remains to be determined.

Postoperative urinary retention after inguinal hernia repair: a single institution experience.

PURPOSE: Inguinal hernia repair is a common general surgery procedure with low morbidity. However, postoperative urinary retention (PUR) occurs in up to 22% of patients, resulting in further extraneous treatments.This single institution series investigates whether patient comorbidities, surgical approaches, and anesthesia methods are associated with developing PUR after inguinal hernia repairs. METHODS: This is a single institution retrospective review of inguinal hernia from 2012 to 2015. PUR was defined as patients without a postoperative urinary catheter who subsequently required bladder decompression due to an inability to void. Univariate and multivariate logistic regressions were performed to quantify the associations between patient, surgical, and anesthetic factors with PUR. Stratification analysis was conducted at age of 50 years. RESULTS: 445 patients were included (42.9% laparoscopic and 57.1% open). Overall rate of PUR was 11.2% (12% laparoscopic, 10.6% open, and p = 0.64). In univariate analysis, PUR was significantly associated with patient age >50 and history of benign prostatic hyperplasia (BPH). Risk stratification for age >50 revealed in this cohort a 2.49 times increased PUR risk with lack of intraoperative bladder decompression (p = 0.013). CONCLUSIONS: At our institution, we found that patient age, history of BPH, and bilateral repair were associated with PUR after inguinal hernia repair. No association was found with PUR and laparoscopic vs open approach. Older males may be at higher risk without intraoperative bladder decompression, and therefore, catheter placement should be considered in this population, regardless of surgical approach.

H-NS mediated compaction of DNA visualised by atomic force microscopy.

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in gene regulation and DNA compaction. To get more insight into the mechanism of DNA compaction we applied atomic force microscopy (AFM) to study the structure of H-NS-DNA complexes. On circular DNA molecules two different levels of H-NS induced condensation were observed. H-NS induced lateral condensation of large regions of the plasmid. In addition, large globular structures were identified that incorporated a considerable amount of DNA. The formation of these globular structures appeared not to be dependent on any specific sequence. On the basis of the AFM images, a model for global condensation of the chromosomal DNA by H-NS is proposed.

DNA bending by photolyase in specific and non-specific complexes studied by atomic force microscopy.

Specific and non-specific complexes of DNA and photolyase are visualised by atomic force microscopy. As a substrate for photolyase a 1150 bp DNA restriction fragment was UV-irradiated to produce damaged sites at random positions. Comparison with a 735 bp undamaged DNA fragment made it possible to separate populations of specific and non-specific photolyase complexes on the 1150 bp fragment, relieving the need for highly defined substrates. Thus it was possible to compare DNA bending for specific and non-specific interactions. Non-specific complexes show no significant bending but increased rigidity compared to naked DNA, whereas specific complexes show DNA bending of on average 36 degrees and higher flexibility. A model obtained by docking shows that photolyase can accommodate a 36 degrees bent DNA in the vicinity of the active site.

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Tel-1 transposon-like elements of Tetrahymena thermophila are associated with micronuclear genome rearrangements.

The micronuclear genome of Tetrahymena thermophila contains Tel-1 elements that structurally resemble transposons. Here we present molecular evidence that Tel-1 transposon-like elements are mobile. The arrangements of Tel-1 elements in the micronuclear genomes of several T. thermophila strains and cell lines were assayed by Southern blotting. The molecular evidence for Tel-1 transposition is most striking in strains that have undergone unusual laboratory-induced meioses. The genetic history of the strains exhibiting evidence of Tel-1 transposition is consistent with periods of genome restructuring in response to genomic "shock" that B. McClintock has suggested could result in transposon activation.

Refining sweet sorghum to ethanol and sugar: economic trade-offs in the context of North China.

Reducing the use of non-renewable fossil energy reserves together with improving the environment are two important reasons that drive interest in the use of bioethanol as an automotive fuel. Conversion of sugar and starch to ethanol has been proven at an industrial scale in Brazil and the United States, respectively, and this alcohol has been able to compete with conventional gasoline due to various incentives. In this paper, we examined making ethanol from the sugar extracted from the juice of sweet sorghum and/or from the hemicellulose and cellulose in the residual sorghum bagasse versus selling the sugar from the juice or burning the bagasse to make electricity in four scenarios in the context of North China. In general terms, the production of ethanol from the hemicellulose and cellulose in bagasse was more favorable than burning it to make power, but the relative merits of making ethanol or sugar from the juice was very sensitive to the price of sugar in China. This result was confirmed by both process economics and analysis of opportunity costs. Thus, a flexible plant capable of making both sugar and fuel-ethanol from the juice is recommended. Overall, ethanol production from sorghum bagasse appears very favorable, but other agricultural residues such as corn stover and rice hulls would likely provide a more attractive feedstock for making ethanol in the medium and long term due to their extensive availability in North China and their independence from other markets. Furthermore, the process for residue conversion was based on particular design assumptions, and other technologies could enhance competitiveness while considerations such as perceived risk could impede applications.

Structural basis for preferential binding of H-NS to curved DNA.

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in transcription regulation and DNA compaction. H-NS exerts its role in DNA condensation by non-specific interactions with DNA. With respect to transcription regulation preferential binding sites in the promoter regions of different genes have been reported. In this paper we describe the analysis of H-NS-DNA complexes on a preferred H-NS binding site by atomic force microscopy. On the basis of these data we present a model for the specific recognition of DNA by H-NS as a function of DNA curvature.

Twenty years of trials, tribulations, and research progress in bioethanol technology: selected key events along the way.

The projected cost of ethanol production from cellulosic biomass has been reduced by almost a factor of four over the last 20 yr. Thus, it is now competitive for blending with gasoline, and several companies are working to build the first plants. However, technology development faced challenges at all levels. Because the benefits of bioethanol were not well understood, it was imperative to clarify and differentiate its attributes. Process engineering was invaluable in focusing on promising opportunities for improvements, particularly in light of budget reductions, and in tracking progress toward a competitive goal. Now it is vital for one or more commercial projects to be successful, and improving our understanding of process fundamentals will reduce the time and costs for commercialization. Additionally, the cost of bioethanol must be cut further to be competitive as a pure fuel in the open market, and aggressive technology advances are required to meet this target.

Heat transfer considerations in design of a batch tube reactor for biomass hydrolysis.

Biologic conversion of inexpensive and abundant sources of cellulosic biomass offers a low-cost route to production of fuels and commodity chemicals that can provide unparalleled environmental, economic, and strategic benefits. However, low-cost, high-yield technologies are needed to recover sugars from the hemicellulose fraction of biomass and to prepare the remaining cellulose fraction for subsequent hydrolysis. Uncatalyzed hemicellulose hydrolysis in flow-through systems offers a number of important advantages for removal of hemicellulose sugars, and it is believed that oligomers could play an important role in explaining why the performance of flow-through systems differs from uncatalyzed steam explosion approaches. Thus, an effort is under way to study oligomer formation kinetics, and a small batch reactor is being applied to capture these important intermediates in a closed system that facilitates material balance closure for varying reaction conditions. In this article, heat transfer for batch tubes is analyzed to derive temperature profiles for different tube diameters and assess the impact on xylan conversion. It was found that the tube diameter must be <0.5 in. for xylan hydrolysis to follow the kinetics expected for a uniform temperature system at typical operating conditions.

Cellulose and hemicellulose hydrolysis models for application to current and novel pretreatment processes.

Acids catalyze the hydrolysis of cellulose and hemicellulose to produce sugars that organisms can ferment to ethanol and other products. However, advanced low- and no-acid technologies are critical if we are to reduce bio-ethanol costs to be competitive as a pure fuel. We believe carbohydrate oligomers play a key role in explaining the performance of such hydrolysis processes and that kinetic models would help us understand their role. Various investigations have developed reaction rate expressions based on an Arrhenius temperature dependence that is first order in substrate concentration and close to first order in acid concentration. In this article, we evaluate these existing hydrolysis models with the goal of providing a foundation for a unified model that can predict performance of both current and novel pretreatment process configurations.

Fluorescence correlation spectroscopy of the binding of nucleotide excision repair protein XPC-hHr23B with DNA substrates.

The interaction of the nucleotide excision repair (NER) protein dimeric complex XPC-hHR23B, which is implicated in the DNA damage recognition step, with three Cy3.5 labeled 90-bp double-stranded DNA substrates (unmodified, with a central unpaired region, and cholesterol modified) and a 90-mer single-strand DNA was investigated in solution by fluorescence correlation spectroscopy. Autocorrelation functions obtained in the presence of an excess of protein show larger diffusion times (tau (d)) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (theta), as a way to describe the percentage of protein bound to DNA, was directly estimated from FCS data. A significantly stronger binding capability for the cholesterol modified substrate (78% DNA bound) than for other double-stranded DNA substrates was observed, while the lowest affinity was found for the single-stranded DNA (27%). This is in accordance with a damage recognition role of the XPC protein. The similar affinity of XPC for undamaged and 'bubble' DNA substrates (58% and 55%, respectively) indicates that XPC does not specifically bind to this type of DNA substrate comprising a large (30-nt) central unpaired region.

Nanoparticle-induced fluorescence lifetime modification as nanoscopic ruler: demonstration at the single molecule level.

We combine interferometric detection of single gold nanoparticles, single molecule microscopy, and fluorescence lifetime measurement to study the modification of the fluorescence decay rate of an emitter close to a nanoparticle. In our experiment, gold particles with a diameter of 15 nm were attached to single dye molecules via double-stranded DNA of different lengths. Nanoparticle-induced lifetime modification (NPILM) has promise in serving as a nanoscopic ruler for the distance range well beyond 10 nm, which is the upper limit of fluorescence resonant energy transfer (FRET). Furthermore, the simultaneous detection of single nanoparticles and fluorescent molecules presented in this work provides new opportunities for single molecule biophysical studies.