Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase.

Mutations in human (Homo sapiens) ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized in part by brain lesions, lactic acidemia, excretion of ethylmalonic acid, and ultimately death. ETHE1-like genes are found in a wide range of organisms; however, the biochemical and physiological role(s) of ETHE1 have not been examined outside the context of ethylmalonic encephalopathy. In this study we characterized Arabidopsis (Arabidopsis thaliana) ETHE1 and determined the effect of an ETHE1 loss-of-function mutation to investigate the role(s) of ETHE1 in plants. Arabidopsis ETHE1 is localized in the mitochondrion and exhibits sulfur dioxygenase activity. Seeds homozygous for a DNA insertion in ETHE1 exhibit alterations in endosperm development that are accompanied by a delay in embryo development followed by embryo arrest by early heart stage. Strong ETHE1 labeling was observed in the peripheral and chalazal endosperm of wild-type seeds prior to cellularization. Therefore, ETHE1 appears to play an essential role in regulating sulfide levels in seeds.

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Human glyoxalase II (Glx2) was over-expressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogs were characterized using metal analyses, steady-state and pre-steady state kinetics, and NMR and EPR spectroscopies in order to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly one equivalents each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analog containing two equivalents of Zn(II) cannot be prepared. EPR studies suggest that most of the iron in recombinant Glx2 is Fe(II). NMR studies show that Fe(II) binds to the consensus Zn2 site in Glx2 and that this site can also bind Co(II) and Ni(II), suggesting that Zn(II) binds to the consensus Zn1 site. The NMR studies also reveal the presence of a dinuclear Co(II) center in Co(II)-substituted Glx2. Steady-state and pre-steady state kinetic studies show that Glx2 containing only one equivalent of Zn(II) is catalytically-active and that the metal ion in the consensus Zn2 site has little effect on catalytic activity. Taken together, these studies suggest that Glx2 contains a Fe(II)Zn(II) center in vivo, but that the catalytic activity is due to Zn(II) in the Zn1 site.

Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically-active human GLX2. The resulting enzymes were over-expressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis. The R246H/N248Y double mutant exhibited low level Slactoylglutathione hydrolase activity, while the R246H/N248Y/Q325R/R328K mutant exhibited a 1.5-to 2-fold increase in k cat and a decrease in K m as compared to the values exhibited by the double mutant. In contrast the R246H mutant of GLX2-1 did not exhibit glyoxalase 2 activity. Zn(II)-loaded R246H GLX2-1 enzyme bound 2 equivalents of Zn(II), and 1 H NMR spectra of the Co(II)-substituted analog of this enzyme strongly suggests that the introduced histidine binds to Co(II). EPR studies indicate the presence of significant amounts a dinuclear metal ion-containing center. Therefore, an active GLX2 enzyme requires both the presence of a properly-positioned metal center and significant non-metal, enzyme-substrate contacts, with tyrosine 255 being particularly important.The glyoxalase system consists of two enzymes, lactoylglutathione lyase (GLX1) and hydroxyacylglutathione hydrolase (GLX2). GLX1 is capable of forming S-(2-hydroxyacyl) glutathione (SLG), which is produced from a thiohemiacetal that is formed from the spontaneous reaction of methylglyoxal with glutathione. SLG is then hydrolyzed by GLX2 to produce lactate and glutathione. GLX1 can utilize a number of α-ketoaldehydes; however, methylglyoxal (MG), a cytotoxic and mutagenic compound formed primarily as a byproduct of carbohydrate and lipid metabolism and from triose phosphates, is thought to be the primary physiological substrate of the system(1-5). SLG can also be metabolized by γ-glutamyltransferase and dipeptidase, which generate N-D-lactoylcysteine that passes from cell to cell and can inhibit nucleotide synthesis(6) and ultimately DNA synthesis (7). Therefore, the glyoxalase system, which depletes MG and SLG, is critical for cellular detoxification in aerobic organisms (6).

With developing understanding that host-associated microbiota play significant roles in individual health and fitness, taking an interdisciplinary approach combining microbiome research with conservation science is increasingly favored. Here we establish the scat microbiome of the imperiled Channel Island fox (Urocyon littoralis) and examine the effects of geography and captivity on the variation in bacterial communities. Using high throughput 16S rRNA gene amplicon sequencing, we discovered distinct bacterial communities in each island fox subspecies. Weight, timing of the sample collection, and sex contributed to the geographic patterns. We uncovered significant taxonomic differences and an overall decrease in bacterial diversity in captive versus wild foxes. Understanding the drivers of microbial variation in this system provides a valuable lens through which to evaluate the health and conservation of these genetically depauperate foxes. The island-specific bacterial community baselines established in this study can make monitoring island fox health easier and understanding the implications of inter-island translocation clearer. The decrease in bacterial diversity within captive foxes could lead to losses in the functional services normally provided by commensal microbes and suggests that zoos and captive breeding programs would benefit from maintaining microbial diversity.

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