Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

Plastic Food Packaging from Five Countries Contains Endocrine- and Metabolism-Disrupting Chemicals.

Plastics are complex chemical mixtures of polymers and various intentionally and nonintentionally added substances. Despite the well-established links between certain plastic chemicals (bisphenols and phthalates) and adverse health effects, the composition and toxicity of real-world mixtures of plastic chemicals are not well understood. To assess both, we analyzed the chemicals from 36 plastic food contact articles from five countries using nontarget high-resolution mass spectrometry and reporter-gene assays for four nuclear receptors that represent key components of the endocrine and metabolic system. We found that chemicals activating the pregnane X receptor (PXR), peroxisome proliferator receptor γ (PPARγ), estrogen receptor α (ERα), and inhibiting the androgen receptor (AR) are prevalent in plastic packaging. We detected up to 9936 chemical features in a single product and found that each product had a rather unique chemical fingerprint. To tackle this chemical complexity, we used stepwise partial least-squares regressions and prioritized and tentatively identified the chemical features associated with receptor activity. Our findings demonstrate that most plastic food packaging contains endocrine- and metabolism-disrupting chemicals. Since samples with fewer chemical features induce less toxicity, chemical simplification is key to producing safer plastic packaging.

Beyond the Nucleus: Plastic Chemicals Activate G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are central mediators of cell signaling and physiological function. Despite their biological significance, GPCRs have not been widely studied in the field of toxicology. Herein, we investigated these receptors as novel targets of plastic chemicals using a high-throughput drug screening assay with 126 human non-olfactory GPCRs. In a first-pass screen, we tested the activity of triphenol phosphate, bisphenol A, and diethyl phthalate, as well as three real-world mixtures of chemicals extracted from plastic food packaging covering all major polymer types. We found 11 GPCR-chemical interactions, of which the chemical mixtures exhibited the most robust activity at adenosine receptor 1 (ADORA1) and melatonin receptor 1 (MTNR1A). We further confirm that polyvinyl chloride and polyurethane products contain ADORA1 or MTNRA1 agonists using a confirmatory secondary screen and pharmacological knockdown experiments. Finally, an analysis of the associated gene ontology terms suggests that ADORA1 and MTNR1A activation may be linked to downstream effects on circadian and metabolic processes. This work highlights that signaling disruption caused by plastic chemicals is broader than that previously believed and demonstrates the relevance of nongenomic pathways, which have, thus far, remained unexplored.

Exposure to microplastics affects fatty acid composition in the Japanese quail depending on sex and particle size.

Plastic ingestion in birds is a widespread phenomenon of increasing concern. However, little is known about how exposure to microplastics (MP) affects the health of birds. In other organisms, MP exposure alters lipid metabolism and composition. If also true in birds, this could affect their fitness, especially since birds heavily rely on lipids during migration and egg production. Therefore, the aim of this study was to determine if ingestion of MP (polypropylene and polyethylene collected in nature) in two size ranges, large (3 mm) and small (<125 µm), affects lipid metabolism in the Japanese quail (Coturnix japonica). We orally exposed 55 one-week-old quail during 5 weeks to a total of 600 mg of MP in sizes of either large, small, or a mixture of both. After the exposure period, females fed small MP had higher liver masses compared to control females (on average ± SD, 8.95 ± 2.3 g vs. 6.34 ± 1.0 g), while liver lipid content did not differ in either males or females. The levels of monounsaturated fatty acids were lower in females exposed to large MP and the mixture of both MP sizes compared to controls. Females exposed to MP also had different levels of oleic- (18:1) and palmitoleic (16:1) acid compared to controls dependent on MP size. Exposure to small MP increased levels of palmitic- (16:0) and palmitoleic (16:1) acid in both males and females suggesting a possible increase in de novo fatty acid synthesis. Taken together, our results point towards a sex-specific sensitivity to MP as well as size-dependent MP effects on lipid metabolism in birds. Disruption of fatty acid composition could affect important life stages in female birds, such as migration and egg-laying. We stress the importance of further research focused on determining the mechanisms of action of MP on lipid metabolism.

Integrative omics-analysis of lipid metabolism regulation by peroxisome proliferator-activated receptor a and b agonists in male Atlantic cod.

Lipid metabolism is essential in maintaining energy homeostasis in multicellular organisms. In vertebrates, the peroxisome proliferator-activated receptors (PPARs, NR1C) regulate the expression of many genes involved in these processes. Atlantic cod (Gadus morhua) is an important fish species in the North Atlantic ecosystem and in human nutrition, with a highly fatty liver. Here we study the involvement of Atlantic cod Ppar a and b subtypes in systemic regulation of lipid metabolism using two model agonists after in vivo exposure. WY-14,643, a specific PPARA ligand in mammals, activated cod Ppara1 and Ppara2 in vitro. In vivo, WY-14,643 caused a shift in lipid transport both at transcriptional and translational level in cod. However, WY-14,643 induced fewer genes in the fatty acid beta-oxidation pathway compared to that observed in rodents. Although GW501516 serves as a specific PPARB/D ligand in mammals, this compound activated cod Ppara1 and Ppara2 as well as Pparb in vitro. In vivo, it further induced transcription of Ppar target genes and caused changes in lipid composition of liver and plasma. The integrative approach provide a foundation for understanding how Ppars are engaged in regulating lipid metabolism in Atlantic cod physiology. We have shown that WY-14,643 and GW501516 activate Atlantic cod Ppara and Pparb, affect genes in lipid metabolism pathways, and induce changes in the lipid composition in plasma and liver microsomal membranes. Particularly, the combined transcriptomic, proteomics and lipidomics analyses revealed that effects of WY-14,643 on lipid metabolism are similar to what is known in mammalian studies, suggesting conservation of Ppara functions in mediating lipid metabolic processes in fish. The alterations in the lipid profiles observed after Ppar agonist exposure suggest that other chemicals with similar Ppar receptor affinities may cause disturbances in the lipid regulation of fish. Model organism: Atlantic cod (Gadus morhua). LSID: urn:lsid:zoobank.org:act:389BE401-2718-4CF2-BBAE-2E13A97A5E7B. COL Identifier: 6K72F.

SMUG1 regulates fat homeostasis leading to a fatty liver phenotype in mice.

Fatty liver diseases are a major health threat across the western world, leading to cirrhosis and premature morbidity and mortality. Recently, a correlation between the base excision repair enzyme SMUG1 and metabolic homeostasis was identified. As the molecular mechanisms remain unknown, we exploited a SMUG1-knockout mouse model to gain insights into this association by characterizing the liver phenotype in young vs old SMUG1-null mice. We observed increased weight and fat content in one-year old animals, with altered activity of enzymes important for fatty acids influx and uptake. Consistently, lipidomic profiling showed accumulation of free fatty acids and triglycerides in SMUG1-null livers. Old SMUG1-knockout mice also displayed increased hepatocyte senescence and DNA damage at telomeres. Interestingly, RNA sequencing revealed widespread changes in the expression of lipid metabolic genes already in three months old animals. In summary, SMUG1 modulates fat metabolism favouring net lipogenesis and resulting in development of a fatty liver phenotype.

Overexpression of Two New Acyl-CoA:Diacylglycerol Acyltransferase 2-Like Acyl-CoA:Sterol Acyltransferases Enhanced Squalene Accumulation in Aurantiochytrium limacinum.

Thraustochytrids are heterotrophic marine eukaryotes known to accumulate large amounts of triacylglycerols, and they also synthesize terpenoids like carotenoids and squalene, which all have an increasing market demand. However, a more extensive knowledge of the lipid metabolism is needed to develop thraustochytrids for profitable biomanufacturing. In this study, two putative type-2 Acyl-CoA:diacylglycerol acyltransferases (DGAT2) genes of Aurantiochytrium sp. T66, T66ASATa, and T66ASATb, and their homologs in Aurantiochytrium limacinum SR21, AlASATa and AlASATb, were characterized. In A. limacinum SR21, genomic knockout of AlASATb reduced the amount of the steryl esters of palmitic acid, SE (16:0), and docosahexaenoic acid, SE (22:6). The double mutant of AlASATa and AlASATb produced even less of these steryl esters. The expression and overexpression of T66ASATb and AlASATb, respectively, enhanced SE (16:0) and SE (22:6) production more significantly than those of T66ASATa and AlASATa. In contrast, these mutations did not significantly change the level of triacylglycerols or other lipid classes. The results suggest that the four genes encoded proteins possessing acyl-CoA:sterol acyltransferase (ASAT) activity synthesizing both SE (16:0) and SE (22:6), but with the contribution from AlASATb and T66ASATb being more important than that of AlASATa and T66ASATa. Furthermore, the expression and overexpression of T66ASATb and AlASATb enhanced squalene accumulation in SR21 by up to 88%. The discovery highlights the functional diversity of DGAT2-like proteins and provides valuable information on steryl ester and squalene synthesis in thraustochytrids, paving the way to enhance squalene production through metabolic engineering.

THESEUS1 modulates cell wall stiffness and abscisic acid production in Arabidopsis thaliana.

Plant cells can be distinguished from animal cells by their cell walls and high-turgor pressure. Although changes in turgor and the stiffness of cell walls seem coordinated, we know little about the mechanism responsible for coordination. Evidence has accumulated that plants, like yeast, have a dedicated cell wall integrity maintenance mechanism. It monitors the functional integrity of the wall and maintains integrity through adaptive responses induced by cell wall damage arising during growth, development, and interactions with the environment. These adaptive responses include osmosensitive induction of phytohormone production, defense responses, as well as changes in cell wall composition and structure. Here, we investigate how the cell wall integrity maintenance mechanism coordinates changes in cell wall stiffness and turgor in Arabidopsis thaliana We show that the production of abscisic acid (ABA), the phytohormone-modulating turgor pressure, and responses to drought depend on the presence of a functional cell wall. We find that the cell wall integrity sensor THESEUS1 modulates mechanical properties of walls, turgor loss point, ABA biosynthesis, and ABA-controlled processes. We identify RECEPTOR-LIKE PROTEIN 12 as a component of cell wall integrity maintenance-controlling, cell wall damage-induced jasmonic acid (JA) production. We propose that THE1 is responsible for coordinating changes in turgor pressure and cell wall stiffness.

Plastic Products Leach Chemicals That Induce In Vitro Toxicity under Realistic Use Conditions.

Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.

Combined Metabolome and Lipidome Analyses for In-Depth Characterization of Lipid Accumulation in the DHA Producing Aurantiochytrium sp. T66.

Thraustochytrids are marine heterotrophic microorganisms known for their potential to accumulate docosahexaenoic acid (DHA)-enriched lipids. There have been many attempts to improve thraustochytrid DHA bioprocesses, especially through traditional optimization of cultivation and media conditions. Nevertheless, thraustochytrid-based bioprocesses are still not commercially competitive for high volume-low cost production of DHA. Thus, it is realized that genetic and metabolic engineering strategies are needed for the development of commercially competitive thraustochytrid DHA cell factories. Here, we present an analytical workflow for high resolution phenotyping at metabolite and lipid levels to generate deeper insight into the thraustochytrid physiology, with particular focus on central carbon and redox metabolism. We use time-series sampling during unlimited growth and nitrogen depleted triggering of DHA synthesis and lipid accumulation (LA) to show-case our methodology. The mass spectrometric absolute quantitative metabolite profiling covered glycolytic, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) metabolites, amino acids, complete (deoxy)nucleoside phosphate pools, CoA and NAD metabolites, while semiquantitative high-resolution supercritical fluid chromatography MS/MS was applied for the lipid profiling. Interestingly, trace amounts of a triacylglycerols (TG) with DHA incorporated in all three acyl positions was detected, while TGs 16:0_16:0_22:6 and 16:0_22:6_22:6 were among the dominant lipid species. The metabolite profiling data indicated that lipid accumulation is not limited by availability of the acyl chain carbon precursor acetyl-CoA nor reducing power (NADPH) but rather points to the TG head group precursor glycerol-3-phosphate as the potential cause at the metabolite level for the gradual decline in lipid production throughout the cultivation. This high-resolution phenotyping provides new knowledge of changes in the central metabolism during growth and LA in thraustochytrids and will guide target selection for metabolic engineering needed for further improvements of this DHA cell factory.

Diet and Life Stage-Associated Lipidome Remodeling in Atlantic Salmon.

Salmon is an important source of long-chain highly unsaturated fatty acids (LC-HUFAs) such as 22:6n-3 [docosahexaenoic acid (DHA)]. In the present study, we conducted two identical experiments on salmon in freshwater (FW) and seawater (SW) stages, with a diet switch from fish oil (high in LC-HUFA) to vegetable oil (low in LC-HUFA) and vice versa. Our aim was to investigate the diet and life stage-specific features of lipid uptake (gut), processing (liver), and deposition (muscle). The lipid composition changed much faster in the gut of SW fish relative to FW fish, suggesting that the former had a higher rate of lipid absorption and transport. SW fish also had higher expression of phospholipid synthesis and lipoprotein formation genes in the gut, whereas FW fish had higher expression of lipid synthesis genes in the liver. All phospholipids except PC-44:12 and PE-44:12 were less abundant in SW, suggesting that SW fish have a higher requirement for DHA.

High Throughput Semiquantitative UHPSFC-MS/MS Lipid Profiling and Lipid Class Determination.

High throughput and high-resolution lipid analyses are important for many biological model systems and research questions. This comprises both monitoring at the individual lipid species level and broad lipid classes. Here, we present a nontarget semiquantitative lipidomics workflow based on ultrahigh performance supercritical fluid chromatography (UHPSFC)-mass spectrometry (MS). The optimized chromatographic conditions enable the base-line separation of both nonpolar and polar classes in a single 7-minute run. Ionization efficiencies of lipid classes vary 10folds in magnitude and great care must be taken in a direct interpretation of raw data. Therefore, the inclusion of internal standards or experimentally determined Response factors (RF) are highly recommended for the conversion of raw abundances into (semi) quantitative data. We have deliberately developed an algorithm for automatic semiquantification of lipid classes by RF. The workflow was tested and validated using a bovine liver extract with satisfactory results. The RF corrected data provide a more representative relative lipid class determination, but also the interpretation of individual lipid species should be performed on RF corrected data. In addition, semiquantification can be improved by using internal or also external standards when more accurate quantitative data are of interest but this requires validation for all new sample types. The workflow established greatly extends the potential of nontarget UHPSFC-MS/MS based analysis.

Impaired NDRG1 functions in Schwann cells cause demyelinating neuropathy in a dog model of Charcot-Marie-Tooth type 4D.

Mutations in the N-myc downstream-regulated gene 1 (NDRG1) cause degenerative polyneuropathy in ways that are poorly understood. We have investigated Alaskan Malamute dogs with neuropathy caused by a missense mutation in NDRG1. In affected animals, nerve levels of NDRG1 protein were reduced by more than 70% (p< 0.03). Nerve fibers were thinly myelinated, loss of large myelinated fibers was pronounced and teased fiber preparations showed both demyelination and remyelination. Inclusions of filamentous material containing actin were present in adaxonal Schwann cell cytoplasm and Schmidt-Lanterman clefts. This condition strongly resembles the human Charcot-Marie-Tooth type 4D. However, the focally folded myelin with adaxonal infoldings segregating the axon found in this study are ultrastructural changes not described in the human disease. Furthermore, lipidomic analysis revealed a profound loss of peripheral nerve lipids. Our data suggest that the low levels of mutant NDRG1 is insufficient to support Schwann cells in maintaining myelin homeostasis.

Quantitative transcriptomics, and lipidomics in evaluating ovarian developmental effects in Atlantic cod (Gadus morhua) caged at a capped marine waste disposal site.

In the present study, a previously capped waste disposal site at Kollevåg (Norway) was selected to study the effects of contaminant leakage on biomarkers associated with Atlantic cod (Gadus morhua) reproductive endocrinology and development. Immature cod were caged for 6 weeks at 3 locations, selected to achieve a spatial gradient of contamination, and compared to a reference station. Quantitative transcriptomic, and lipidomic analysis was used to evaluate the effects of the potential complex contaminant mixture on ovarian developmental and endocrine physiology. The number of expressed transcripts, with 0.75 log2-fold differential expression or more, varied among stations and paralleled the severity of contamination. Particularly, significant bioaccumulation of ∑PCB-7, ∑DDTs and ∑PBDEs were observed at station 1, compared to the other station, including the reference station. Respectively 1416, 698 and 719 differentially expressed genes (DEGs), were observed at stations 1, 2 and 3, compared to the reference station, with transcripts belonging to steroid hormone synthesis pathway being significantly upregulation. Transcription factors such as esr2 and ahr2 were increased at all three stations, with highest fold-change at Station 1. MetaCore pathway maps identified affected pathways that are involved in ovarian physiology, where some unique pathways were significantly affected at each station. For the lipidomics, sphingolipid metabolism was particularly affected at station 1, and these effects paralleled the high contaminant burden at this station. Overall, our findings showed a novel and direct association between contaminant burden and ovarian toxicological and endocrine physiological responses in cod caged at the capped Kollevåg waste disposal site.

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems.

Absolute quantification of intracellular metabolite pools is a prerequisite for modeling and in-depth biological interpretation of metabolomics data. It is the final step of an elaborate metabolomics workflow, with challenges associated with all steps-from sampling to quantifying the physicochemically diverse metabolite pool. Chromatographic separation combined with mass spectrometric (MS) detection is the superior platform for high coverage, selective, and sensitive detection of metabolites. Herein, we apply our quantitative MS-metabolomics workflow to measure and present the central carbon metabolome of a panel of commonly applied biological model systems. The workflow includes three chromatographic methods combined with isotope dilution tandem mass spectrometry to allow for absolute quantification of 68 metabolites of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and the amino acid and (deoxy) nucleoside pools. The biological model systems; Bacillus subtilis, Saccharomyces cerevisiae, two microalgal species, and four human cell lines were all cultured in commonly applied culture media and sampled in exponential growth phase. Both literature and databases are scarce with comprehensive metabolite datasets, and existing entries range over several orders of magnitude. The workflow and metabolite panel presented herein can be employed to expand the list of reference metabolomes, as encouraged by the metabolomics community, in a continued effort to develop and refine high-quality quantitative metabolomics workflows.

Demyelinating polyneuropathy in goats lacking prion protein.

Studies in mice with ablation of Prnp, the gene that encodes the cellular prion protein (PrPC ), have led to the hypothesis that PrPC is important for peripheral nerve myelin maintenance. Here, we have used a nontransgenic animal model to put this idea to the test; namely, goats that, due to a naturally occurring nonsense mutation, lack PrPC . Teased nerve fiber preparation revealed a demyelinating pathology in goats without PrPC . Affected nerves were invaded by macrophages and T cells and displayed vacuolated fibers, shrunken axons, and onion bulbs. Peripheral nerve lipid composition was similar in young goats with or without PrPC , but markedly different between corresponding groups of adult goats, reflecting the progressive nature of the neuropathy. This is the first report of a subclinical demyelinating polyneuropathy caused by loss of PrPC function in a nontransgenic mammal.

A versatile scaffold contributes to damage survival via sumoylation and nuclease interactions.

DNA repair scaffolds mediate specific DNA and protein interactions in order to assist repair enzymes in recognizing and removing damaged sequences. Many scaffold proteins are dedicated to repairing a particular type of lesion. Here, we show that the budding yeast Saw1 scaffold is more versatile. It helps cells cope with base lesions and protein-DNA adducts through its known function of recruiting the Rad1-Rad10 nuclease to DNA. In addition, it promotes UV survival via a mechanism mediated by its sumoylation. Saw1 sumoylation favors its interaction with another nuclease Slx1-Slx4, and this SUMO-mediated role is genetically separable from two main UV lesion repair processes. These effects of Saw1 and its sumoylation suggest that Saw1 is a multifunctional scaffold that can facilitate diverse types of DNA repair through its modification and nuclease interactions.

Nucleases in homologous recombination as targets for cancer therapy.

Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.

HPLC-ED of low-molecular weight brominated phenols and tetrabromobisphenol A using pretreated carbon fiber microelectrode.

Electrochemically pretreated carbon fiber microelectrode was used to develop a simple, fast and sensitive HPLC-ECD method for the determination of brominated phenols. In addition to simple mono-, di- and tri-bromophenols (4-bromophenol, 2,4-dibromophenol, 2,6-dibromophenol, 2,4,6-tri-bromophenol) the possibility of electrochemical detection of 3,3',5,5'-tetrabromobisphenol A in oxidation mode is reported for the first time. The isocratic separation was achieved within 14 min using ternary mobile phase consisting of 50mM-phosphate buffer (pH 3.5), acetonitrile and methanol (35/15/50, v/v), and detection potential of E=+1450 mV (vs. Ag/AgCl). The carbon fiber microelectrode permitted to use high anodic potentials (up to +1800 mV vs. Ag/AgCl), the optimum analytical response was achieved at +1450 mV vs. Ag/AgCl. The limits of detection (LOD) for the studied analytes were within the range of 1.8-56.6 ng mL(-1). The developed method was applied to determination of brominated phenols in spiked water samples. Furthermore, after simple extraction with methyl tert-butyl ether, it was possible to quantify tetrabromobisphenol A (TBBA) in a piece of CRT monitor plastic casing. The found amount of TBBA was 10.22 mg kg(-1) (±0.43).

Sumoylation of the Rad1 nuclease promotes DNA repair and regulates its DNA association.

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.