Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 µM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.

Pleiotropic Effects of Caffeine Leading to Chromosome Instability and Cytotoxicity in Eukaryotic Microorganisms.

Caffeine, a methylxanthine analog of purine bases, is a compound that is largely consumed in beverages and medications for psychoactive and diuretic effects and plays many beneficial roles in neuronal stimulation and enhancement of anti-tumor immune responses by blocking adenosine receptors in higher organisms. In single-cell eukaryotes, however, caffeine somehow impairs cellular fitness by compromising cell wall integrity, inhibiting target of rapamycin (TOR) signaling and growth, and overriding cell cycle arrest caused by DNA damage. Among its multiple inhibitory targets, caffeine specifically interacts with phosphatidylinositol 3-kinase (PI3K)-related kinases causing radiosensitization and cytotoxicity via specialized intermediate molecules. Caffeine potentiates the lethality of cells in conjunction with several other stressors such as oxidants, irradiation, and various toxic compounds through largely unknown mechanisms. In this review, recent findings on caffeine effects and cellular detoxification schemes are highlighted and discussed with an emphasis on the inhibitory interactions between caffeine and its multiple targets in eukaryotic microorganisms such as budding and fission yeasts.

Effects of pomegranate peel extract on ruminal and post-ruminal in vitro degradation of rumen inoculum of the dairy cow.

This experiment was carried out to study the effect of water extracted pomegranate peel extract (PE) on ruminal protein degradation and post-ruminal digestion in the dairy cow. PE was added at six levels of total phenolics (g/kg of the basal diet); 3.75 (PE1); 4.4 (PE2); 5.05 (PE3); 5.70 (PE4); and 6.35 (PE5). Rumen degradable crude protein (rdCP) decreased with PE addition (L < 0.0001), but total CP degradability (tdCP) was not affected. Compared to PE0, PE2, and PE3 diets showed higher (L = 0.054, Q = 0.029) digestibility of bypass CP (dBCP). Increasing levels of PE resulted in a decrease in proteolytic bacteria numbers (p < 0.0001). At PE4 and PE5 levels, total VFA and acetate concentrations linearly decreased compared to PE0. PE inclusion lowered the acetate:propionate ratio (L = 0.0001) and Ammonia-N production after 24 h (L = 0.0008) of incubation. The total number of protozoa, genera Dasytricha and Isotricha, and subfamilies Entodiniinae, Diplodiniinae, and Ophrioscolecinae decreased with increasing dietary PE concentration (p < 0.0001). The results suggest that all levels of PE addition reduce the protozoal population and Ammonia-N concentration. All PE levels slowed down protein degradation in the rumen but PE2 and PE3 showed the greatest effect.

Green biosynthesis of silver nanoparticles by Aspergillus niger and its antiamoebic effect against Allovahlkampfia spelaea trophozoite and cyst.

BACKGROUND: Fungi represent an interesting candidate for the synthesis of nanoparticles. The biosynthesis of silver nanoparticles (AgNPs) has many industrial and biomedical indications. We aimed in this work to biologically synthesize silver nanoparticles using Aspergillus niger and to evaluate its effect against the newly identified Allovahlkampfia spelaea that causes resistant human keratitis. MATERIAL AND METHODS: Aspergillus niger (soil isolate) was treated with silver nitrate to produce silver nanoparticles. AgNPs were characterized by Ultraviolet-Visible Spectroscopy, Transmission Electron Microscopy, and Fourier Transform Infrared Spectroscopy. The effect of the synthesized nanoparticles against Allovahlkampfia spelaea growth, encystation, excystation, and toxicity in host cells was evaluated. RESULTS: AgNPs exhibited significant inhibition of Allovahlkampfia spelaea viability and growth of both trophozoites and cysts, with a reduction of amoebic cytotoxic activity in host cells. CONCLUSION: AgNPs may give a promising future to the treatment of Allovahlkampfia spelaea infections in humans.

Carbon limitation in hypereutrophic, periphytic algal wastewater treatment systems.

Global eutrophication degrades water quality in freshwater ecosystems and limits the availability of freshwater for human consumption. While current wastewater treatment facilities (WWTF) remove pathogens and pollutants, many US WWTF continue to discharge nutrients that contribute to eutrophication. Traditional nutrient removal technologies can effectively reduce eutrophication risk, but can have unintended negative consequences on human and environmental health. Alternatives, such as algae-based treatment systems, improve the sustainability of the nutrient recovery process by producing biomass that can be converted to biofuel. However, research is needed to increase the productivity of algal treatment systems to improve their economic viability. Because algae in wastewater treatment systems are grown in wastewater rich in nutrients, the algae could become limited by dissolved inorganic carbon. This hypothesis was tested in 1.2 m long recirculating floways (n = 8 for each treatment/control) by quantifying algal dry mass and wastewater nutrient concentrations in 3 independent experiments: (1) carbon dioxide gas infused vs air infused control; (2) hydrochloric acid acidified vs neutralized solution control vs no chemical addition control; and (3) sodium bicarbonate addition vs no chemical control. Results showed increases in algal biomass after 18 days in wastewater augmented with dissolved inorganic carbon (carbon dioxide or sodium bicarbonate). In contrast, maintaining wastewater at near neutral pH with hydrochloric acid reduced algal productivity relative to controls. Nutrient reductions generally paralleled algal biomass increases except in the bicarbonate addition experiment. These findings provide evidence for the importance of carbon limitation in algal wastewater treatment floways. These results could help explain why carbon dioxide infusions stimulate algae in treatment systems. Furthermore, these results suggest that algae in nutrient enriched, sun-exposed streams (e.g., agricultural ditches or urbanized streams) may become carbon limited during peak periods of productivity. These findings could have important implications for ecosystems undergoing eutrophication as atmospheric carbon dioxide concentrations continue to rise.

Exaptive Evolution of Target of Rapamycin Signaling in Multicellular Eukaryotes.

Target of rapamycin (TOR) is a protein kinase that coordinates metabolism with nutrient and energy availability in eukaryotes. TOR and its primary interactors, RAPTOR and LST8, have been remarkably evolutionarily static since they arose in the unicellular last common ancestor of plants, fungi, and animals, but the upstream regulatory mechanisms and downstream effectors of TOR signaling have evolved considerable diversity in these separate lineages. Here, I focus on the roles of exaptation and adaptation in the evolution of novel signaling axes in the TOR network in multicellular eukaryotes, concentrating especially on amino acid sensing, cell-cell signaling, and cell differentiation.

High-Density pH-Auxostat Fed-Batch Culture of Schizochytrium limacinum SR21 with Acetic Acid as a Carbon Source.

Schizochytrium limacinum SR21 is an important strain for industrial production of docosahexaenoic acid (DHA), which is an important omega-3 fatty acid used in the nutraceutical and food industry. However, the high cost of carbon sources has limited its further application in the market with much larger volume, such as animal feed for aquaculture, poultry, and livestock. To seek low-cost carbon source, acetic acid is tested in the present study. The effect of different factors, including initial carbon source concentration, pH, aeration rate, and nitrogen sources, on biomass, lipid, and DHA production were tested. With optimized culture conditions, the biomass concentration of 146 g/L, total fatty acids (TFAs) of 82.3 g/L, and DHA content of 23.0 g/L were achieved with a pH-auxostat fed-batch cultivation. These results suggested that acetic acid is a promising feedstock for the low-cost production of DHA. Graphical Abstract.

Who is where in the Plastisphere, and why does it matter?

To fully understand how plastic is affecting the ocean, we need to understand how marine life interacts directly with it. Besides their ecological relevance, microbes can affect the distribution, degradation and transfer of plastics to the rest of the marine food web. From amplicon sequencing and scanning electron microscopy, we know that a diverse array of microorganisms rapidly associate with plastic marine debris in the form of biofouling and biofilms, also known as the "Plastisphere." However, observation of multiple microbial interactions in situ, at small spatial scales in the Plastisphere, has been a challenge. In this issue of Molecular Ecology Resources, Schlundt et al. apply the combination labelling and spectral imaging - fluorescence in situ hybridization to study microbial communities on plastic marine debris. The images demonstrate the colocalization of abundant bacterial groups on plastic marine debris at a relatively high taxonomic and spatial resolution while also visualizing biofouling of eukaryotes, such as diatoms and bryozoans. This modern imaging technology provides new possibilities to address questions regarding the ecology of marine microbes on plastic marine debris and describe more specific impacts of plastic pollution in the marine food webs.

Disinfection exhibits systematic impacts on the drinking water microbiome.

Limiting microbial growth during drinking water distribution is achieved either by maintaining a disinfectant residual or through nutrient limitation without using a disinfectant. The impact of these contrasting approaches on the drinking water microbiome is not systematically understood. We use genome-resolved metagenomics to compare the structure, metabolic traits, and population genomes of drinking water microbiome samples from bulk drinking water across multiple full-scale disinfected and non-disinfected drinking water systems. Microbial communities cluster at the structural- and functional potential-level based on the presence/absence of a disinfectant residual. Disinfectant residual alone explained 17 and 6.5% of the variance in structure and functional potential of the drinking water microbiome, respectively, despite including multiple drinking water systems with variable source waters and source water communities and treatment strategies. The drinking water microbiome is structurally and functionally less diverse and variable across disinfected compared to non-disinfected systems. While bacteria were the most abundant domain, archaea and eukaryota were more abundant in non-disinfected and disinfected systems, respectively. Community-level differences in functional potential were driven by enrichment of genes associated with carbon and nitrogen fixation in non-disinfected systems and γ-aminobutyrate metabolism in disinfected systems likely associated with the recycling of amino acids. Genome-level analyses for a subset of phylogenetically-related microorganisms suggests that disinfection selects for microorganisms capable of using fatty acids, presumably from microbial decay products, via the glyoxylate cycle. Overall, we find that disinfection exhibits systematic selective pressures on the drinking water microbiome and may select for microorganisms able to utilize microbial decay products originating from disinfection-inactivated microorganisms. Video abstract.

Molecular Nanomachines Can Destroy Tissue or Kill Multicellular Eukaryotes.

Light-activated molecular nanomachines (MNMs) can be used to drill holes into prokaryotic (bacterial) cell walls and the membrane of eukaryotic cells, including mammalian cancer cells, by their fast rotational movement, leading to cell death. We examined how these MNMs function in multicellular organisms and investigated their use for treatment and eradication of specific diseases by causing damage to certain tissues and small organisms. Three model eukaryotic species, Caenorhabditis elegans, Daphnia pulex, and Mus musculus (mouse), were evaluated. These organisms were exposed to light-activated fast-rotating MNMs and their physiological and pathological changes were studied in detail. Slow rotating MNMs were used to control for the effects of rotation rate. We demonstrate that fast-rotating MNMs caused depigmentation and 70% mortality in C. elegans while reducing the movement as well as heart rate and causing tissue damage in Daphnia. Topically applied light-activated MNMs on mouse skin caused ulceration and microlesions in the epithelial tissue, allowing MNMs to localize into deeper epidermal tissue. Overall, this study shows that the nanomechanical action of light-activated MNMs is effective against multicellular organisms, disrupting cell membranes and damaging tissue in vivo. Customized MNMs that target specific tissues for therapy combined with spatial and temporal control could have broad clinical applications in a variety of benign and malignant disease states including treatment of cancer, parasites, bacteria, and diseased tissues.

Clearing or subverting the enemy: Role of autophagy in protozoan infections.

The protozoan parasites are evolutionarily divergent, unicellular eukaryotic pathogens representing one of the essential sources of parasitic diseases. These parasites significantly affect the economy and cause public health burdens globally. Protozoan parasites share many cellular features and pathways with their respective host cells. This includes autophagy, a process responsible for self-degradation of the cell's components. There is conservation of the central structural and functional machinery for autophagy in most of the eukaryotic phyla, however, Plasmodium and Toxoplasma possess a decreased number of recognizable autophagy-related proteins (ATG). Plasmodium noticeably lacks clear orthologs of the initiating kinase ATG1/ULK1/2, and both Plasmodium and Toxoplasma lack proteins involved in the nucleation of autophagosomes. These organisms have essential apicoplast, a plastid-like non-photosynthetic organelle, which is an adaptation that is used in penetrating the host cell. Furthermore, available evidence suggests that Leishmania, an intracellular protozoan parasite, induces autophagy in macrophages. The autophagic pathway in Trypanosoma cruzi is activated during metacyclogenesis, a process responsible for the infective forms of parasites. Therefore, numerous pathogens have developed strategies to impair the autophagic mechanism in phagocytes. Regulating autophagy is essential to maintain cellular health as adjustments in the autophagy pathway have been linked to the progression of several physiological and pathological conditions in humans. In this review, we report current advances in autophagy in parasites and their host cells, focusing on the ramifications of these studies in the design of potential anti-protozoan therapeutics.

Spatial structure in the "Plastisphere": Molecular resources for imaging microscopic communities on plastic marine debris.

Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the "Plastisphere"-the thin layer of life on the surface of PMD-has been technology-limited. Research into microbe-microbe and microbe-substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI-FISH (combinatorial labelling and spectral imaging - fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha-, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell-count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro-metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales.

Natural mechanisms and artificial PEG-induced mechanism that repair traumatic damage to the plasmalemma in eukaryotes.

Eukaryotic tissues are composed of individual cells surrounded by a plasmalemma that consists of a phospholipid bilayer with hydrophobic heads that bind cell water. Bound-water creates a thermodynamic barrier that impedes the fusion of a plasmalemma with other membrane-bound intracellular structures or with the plasmalemma of adjacent cells. Plasmalemmal damage consisting of small or large holes or complete transections of a cell or axon results in calcium influx at the lesion site. Calcium activates fusogenic pathways that have been phylogenetically conserved and that lower thermodynamic barriers for fusion of membrane-bound structures. Calcium influx also activates phylogenetically conserved sealing mechanisms that mobilize the gradual accumulation and fusion of vesicles/membrane-bound structures that seal the damaged membrane. These naturally occurring sealing mechanisms for different cells vary based on the type of lesion, the type of cell, the proximity of intracellular membranous structures to the lesion and the relation to adjacent cells. The reliability of different measures to assess plasmalemmal sealing need be carefully considered for each cell type. Polyethylene glycol (PEG) bypasses calcium and naturally occurring fusogenic pathways to artificially fuse adjacent cells (PEG-fusion) or artificially seal transected axons (PEG-sealing). PEG-fusion techniques can also be used to rapidly rejoin the closely apposed, open ends of severed axons. PEG-fused axons do not (Wallerian) degenerate and PEG-fused nerve allografts are not immune-rejected, and enable behavioral recoveries not observed for any other clinical treatment. A better understanding of natural and artificial mechanisms that induce membrane fusion should provide better clinical treatment for many disorders involving plasmalemmal damage.

Parasitic Protozoa: Unusual Roles for G-Quadruplexes in Early-Diverging Eukaryotes.

Guanine-quadruplex (G4) motifs, at both the DNA and RNA levels, have assumed an important place in our understanding of the biology of eukaryotes, bacteria and viruses. However, it is generally little known that their very first description, as well as the foundational work on G4s, was performed on protozoans: unicellular life forms that are often parasitic. In this review, we provide a historical perspective on the discovery of G4s, intertwined with their biological significance across the protozoan kingdom. This is a history in three parts: first, a period of discovery including the first characterisation of a G4 motif at the DNA level in ciliates (environmental protozoa); second, a period less dense in publications concerning protozoa, during which DNA G4s were discovered in both humans and viruses; and third, a period of renewed interest in protozoa, including more mechanistic work in ciliates but also in pathogenic protozoa. This last period has opened an exciting prospect of finding new anti-parasitic drugs to interfere with parasite biology, thus adding new compounds to the therapeutic arsenal.

Heavy metal hypertolerant eukaryotic aldehyde dehydrogenase isolated from metal contaminated soil by metatranscriptomics approach.

Constant addition of heavy metal pollutants in soil resulting from anthropogenic activities can prove detrimental to both macro and micro life forms inhabiting the ecosystem. The potential functional roles of eukaryotic microbes in such environment were explored in this study by metatranscriptomics approach. Sized eukaryotic cDNA libraries, library A (<0.5 kb), library B (0.5-1.0 kb), and library C (>1 kb) were constructed from the soil RNA and screened for copper (Cu) tolerance genes by using copper sensitive yeast mutant strain cup1Δ. Screening of the cDNA libraries yielded different clones capable of growing in Cu amended medium. In the present investigation, one of the transcripts PLCc38 from the library C was characterized and tested for its ability to tolerate different heavy metals by using metal sensitive yeast mutants. Sequence analysis PLCc38 showed homology with aldehyde dehydrogenase (ALDH) and capable of tolerating high concentrations of Cu (150-1000 µM). Aldeyde dehydrogenases are stress response enzymes capable of eliminating toxic levels of aldehydes generated due to abiotic environmental stresses. The cDNA PLCc38 also provided tolerance to wide range of Cd (40-100 µM), Zn (10-13 mM) and Co (2-50 mM) concentrations. Oxidative stress tolerance potential of PLCc38 was also confirmed in presence of different concentrations of H2O2. This study proves that PLCc38 is a potent gene associated with metal tolerance which could be used to revegetate heavy metal polluted soil or as a biomarker for detection of metal contamination.

Protist communities are more sensitive to nitrogen fertilization than other microorganisms in diverse agricultural soils.

BACKGROUND: Agricultural food production is at the base of food and fodder, with fertilization having fundamentally and continuously increased crop yield over the last decades. The performance of crops is intimately tied to their microbiome as they together form holobionts. The importance of the microbiome for plant performance is, however, notoriously ignored in agricultural systems as fertilization disconnects the dependency of plants for often plant-beneficial microbial processes. Moreover, we lack a holistic understanding of how fertilization regimes affect the soil microbiome. Here, we examined the effect of a 2-year fertilization regime (no nitrogen fertilization control, nitrogen fertilization, and nitrogen fertilization plus straw amendment) on entire soil microbiomes (bacteria, fungi, and protist) in three common agricultural soil types cropped with maize in two seasons. RESULTS: We found that the application of nitrogen fertilizers more strongly affected protist than bacterial and fungal communities. Nitrogen fertilization indirectly reduced protist diversity through changing abiotic properties and bacterial and fungal communities which differed between soil types and sampling seasons. Nitrogen fertilizer plus straw amendment had greater effects on soil physicochemical properties and microbiome diversity than nitrogen addition alone. Moreover, nitrogen fertilization, even more together with straw, increased soil microbiome network complexity, suggesting that the application of nitrogen fertilizers tightened soil microbiomes interactions. CONCLUSIONS: Together, our results suggest that protists are the most susceptible microbiome component to the application of nitrogen fertilizers. As protist communities also exhibit the strongest seasonal dynamics, they serve as the most sensitive bioindicators of soil changes. Changes in protist communities might have long-term effects if some of the key protist hubs that govern microbiome complexities as top microbiome predators are altered. This study serves as the stepping stone to promote protists as promising agents in targeted microbiome engineering to help in reducing the dependency on exogenous unsustainably high fertilization and pesticide applications.

Antimicrobial Peptides: Amphibian Host Defense Peptides.

Antimicrobial Peptides (AMPs) are one of the most common components of the innate immune system that protect multicellular organisms against microbial invasion. The vast majority of AMPs are isolated from the frog skin. Anuran (frogs and toads) skin contains abundant AMPs that can be developed therapeutically. Such peptides are a unique but diverse group of molecules. In general, more than 50% of the amino acid residues form the hydrophobic part of the molecule. Normally, there are no conserved structural motifs responsible for activity, although the vast majority of the AMPs are cationic due to the presence of multiple lysine residues; this cationicity has a close relationship with antibacterial activity. Notably, recent evidence suggests that synthesis of AMPs in frog skin may confer an advantage on a particular species, although they are not essential for survival. Frog skin AMPs exert potent activity against antibiotic-resistant bacteria, protozoa, yeasts, and fungi by permeating and destroying the plasma membrane and inactivating intracellular targets. Importantly, since they do not bind to a specific receptor, AMPs are less likely to induce resistance mechanisms. Currently, the best known amphibian AMPs are esculentins, brevinins, ranacyclins, ranatuerins, nigrocin-2, magainins, dermaseptins, bombinins, temporins, and japonicins-1 and -2, and palustrin-2. This review focuses on these frog skin AMPs and the mechanisms underlying their antimicrobial activity. We hope that this review will provide further information that will facilitate further study of AMPs and cast new light on novel and safer microbicides.

Short-term impact of biochar amendments on eukaryotic communities in three different soils.

This study determined the loading impacts of wood-based biochar on the eukaryotic community in three different soils (brown sandy loam-BSL, red loam-RL and a black clay loam-BCL) using a pot trial conducted over 10 months. Soil analysis and 18S rRNA gene sequencing performed using the Illumina MiSeq platform was carried out to evaluate the changes in eukaryotic community composition in relation to different added amounts of biochar. It was found that biochar addition had a negligible effect on diversity parameters in the brown sandy loam Kurosol (BSL) and red loam Dermosol (RL) soils. There were, however, significant changes in eukaryotic community composition of these biochar amended soils. These changes were most discernible in the lighter (low clay content) BSL soil for the fungal communities (F = 3.0106, p = 0.0003) present and also when total eukaryotes were considered (F = 2.3907, p = 0.0002). In this respect Glomeromycota seem to be slightly promoted in the lighter BSL soils, which might be due to increased soil porosity and soil chemical fertility. Clay rich BCL soil community structure correlated to a greater degree with soil chemistry influenced by biochar addition. The results showed that soil microeukaryotes were affected by short term carbon amendment, though to a limited extent. The limited effect of biochar loading rates on the soil microbiology could be due to the short incubation period, the lack of added fertiliser nutrients, and also the inherent stability of the soil eukaryotic community. The data suggested the impacts that were observed however included important plant symbiotic organisms. The results also imply biochar applications at different loading levels have differential effects on soil microeurokaryotes in relation to soil properties in particular clay content.

Incomplete base excision repair contributes to cell death from antibiotics and other stresses.

Numerous lethal stresses in bacteria including antibiotics, thymineless death, and MalE-LacZ expression trigger an increase in the production of reactive oxygen species. This results in the oxidation of the nucleotide pool by radicals produced by Fenton chemistry. Following the incorporation of these oxidized nucleotides into the genome, the cell's unsuccessful attempt to repair these lesions through base excision repair (BER) contributes causally to the lethality of these stresses. We review the evidence for this phenomenon of incomplete BER-mediated cell death and discuss how better understanding this pathway could contribute to the development of new antibiotics.

DNA-protein cross-links: Formidable challenges to maintaining genome integrity.

DNA is associated with proteins that are involved in its folding and transaction processes. When cells are exposed to chemical cross-linking agents or free radical-generating ionizing radiation, DNA-associated proteins are covalently trapped within the DNA to produce DNA-protein cross-links (DPCs). DPCs produced by these agents contain cross-linked proteins in an undisrupted DNA strand. Some DNA-metabolizing enzymes that form covalent reaction intermediates can also be irreversibly trapped in the presence of inhibitors or DNA damage to give rise to abortive DPCs. The abortive DPCs often contain cross-linked proteins attached to the 5' or 3' end of a DNA strand break. In vitro studies show that steric hindrance caused by cross-linked proteins impedes the progression of DNA helicases and polymerases and of RNA polymerases. The modes and consequences by which DPCs impede replication and transcription processes are considerably different from those with conventional DNA lesions. Thus, DPCs are formidable challenges to maintaining genome integrity and faithful gene expression. Current models of DPC repair involve direct and indirect removal of DPCs. The direct mechanism works for DPCs that contain topoisomerase 2 attached to the 5' end of DNA. The Mre11-Rad50-Nbs1 complex cleaves the site internal to the DPC and directly releases a DPC-containing oligonucleotide. The indirect mechanism involves degradation of cross-linked proteins by proteasomes or the recently identified DPC proteases Wss1 and Sprtn to relieve steric hindrance of DPCs. The resulting peptide-cross-links might be processed by translesion synthesis or other canonical repair mechanisms: however, the exact mechanism remains to be elucidated.