Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases

Oxidative damage represents a major threat to genomic stability, as the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. In order to prevent these mutagenic events, organisms have evolved GO-DNA glycosylases that remove this oxidized base from DNA. We were interested to find out how GO is processed in the hyperthermophilic archaeon Pyrobaculum aerophilum, which lives at temperatures around 100 degrees C. To this end, we searched its genome for open reading frames (ORFs) bearing the principal hallmark of GO-DNA glycosylases: a helix-hairpin-helix motif and a glycine/proline-rich sequence followed by an absolutely conserved aspartate (HhH-GPD motif). Interestingly, although the P.aerophilum genome encodes three such ORFs, none of these encodes the potent GO-processing activity detected in P.aerophilum extracts. Fractionation of the extracts, followed by analysis of the active fractions by denaturing polyacrylamide gel electrophoresis, showed that the GO-processing enzyme has a molecular size of approximately 30 kDa. Mass spectrometric analysis of proteins in this size range identified several peptides originating from P.aerophilum ORF PAE2237. We now show that PAE2237 encodes AGOG (Archaeal GO-Glycosylase), the founding member of a new family of DNA glycosylases, which can remove GO from single- and double-stranded substrates with great efficienc

Frameshift Mutagenesis and Microsatellite Instability Induced by Human Alkyladenine DNA Glycosylase

Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine, and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than does the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L-induced mutator phenotype. The hAAG-Y127I/H136L and hAAG-Y127I mutants increased the rate of spontaneous frameshifts by up to 120-fold in S. cerevisiae and also induced high rates of microsatellite instability (MSI) in human cells. hAAG and its mutants bind DNA containing one and two base-pair loops with significant affinity, thus shielding them from mismatch repair; the strength of such binding correlates with their ability to induce the mutator phenotype. This study provides important insights into the mechanism of hAAG-induced genomic instability.National Institutes of Health (U.S.) (Grant CA055042)National Institutes of Health (U.S.) (Grant CA115802)National Institutes of Health (U.S.) (Grant ES02109

Development of NILs from heterogeneous inbred families for validating the rust resistance QTL in peanut (Arachis hypogaea L.)

Heterogeneous inbred families segregating for rust resistance were identified from the two crosses involving susceptible (TAG 24 and TG 26) and resistant (GPBD 4) varieties of peanut. Rust-resistant (less than score 5) and rust-susceptible (more than score 5) plants were identified in each HIF and evaluated under rust epiphytotic conditions. The set of plants belonging to the same HIF, but differing significantly in rust resistance, not in other morphological and productivity traits, was regarded as near-isogenic lines (NILs). Largely, rust-resistant NILs had GPBD 4-type allele, and susceptible NILs carried either TAG 24 or TG 26-type allele at the three SSR loci (IPAHM103, GM1536 and GM2301) linked to a major genomic region governing rust resistance. Comparison of the remaining genomic regions between the NILs originating from each of the HIFs using transposon markers indicated a considerably high similarity of 86.4% and 83.1% in TAG 24 × GPPBD 4 and TG 26 × GPBD 4, respectively. These NILs are useful for fine mapping and expression analysis of rust resistance

A Reconfigurable Quantum Local Area Network Over Deployed Fiber

Practical quantum networking architectures are crucial for scaling the connection of quantum resources. Yet quantum network testbeds have thus far underutilized the full capabilities of modern lightwave communications, such as flexible-grid bandwidth allocation. In this work, we implement flex-grid entanglement distribution in a deployed network for the first time, connecting nodes in three distinct campus buildings time-synchronized via the Global Positioning System (GPS). We quantify the quality of the distributed polarization entanglement via log-negativity, which offers a generic metric of link performance in entangled bits per second. After demonstrating successful entanglement distribution for two allocations of our eight dynamically reconfigurable channels, we demonstrate remote state preparation -- the first realization on deployed fiber -- showcasing one possible quantum protocol enabled by the distributed entanglement network. Our results realize an advanced paradigm for managing entanglement resources in quantum networks of ever-increasing complexity and service demands

Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.National Institutes of Health (U.S.) (grant no. P30-ES002109)National Institutes of Health (U.S.) (grant no. GM65337)National Institutes of Health (U.S.) (grant no. GM65337-03S2)National Institutes of Health (U.S.) (grant no. CA055042)National Institutes of Health (U.S.) (grant no. CA092584)Repligen Corporation (KIICR Graduate Fellowship

Higher-Order Assembly of BRCC36-KIAA0157 Is Required for DUB Activity and Biological Function

BRCC36 is a Zn²⁺-dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN⁺ domain protein BRCC36 associates with pseudo DUB MPN⁻ proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher-order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide an explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function

Validation of markers linked to late leaf spot and rust resistance, and selection of superior genotypes among diverse recombinant inbred lines and backcross lines in peanut (Arachis hypogaea L.)

Recombinant inbred lines (RILs) from four populations involving cultivated varieties, and backcross lines from three populations involving cultivated varieties and synthetic tetraploids (developed from wild diploids) were employed for validating late leaf spot (LLS) and rust resistance-linked markers and identifying superior genotypes in peanut. GM2009, GM2301, GM2079, GM1536, GM1954 and IPAHM103 markers showed significant association with rust resistance. They were successfully validated in a new RIL (TG 19 × GPBD 4) and two backcross (DH 86 × ISATGR 278-18 and DH 86 × ISATGR 5) populations. GM1954, GM1009 and GM1573 markers showed significant association with LLS resistance. TAG 19 × GPBD 4 and ICGS 76 × ISATGR 278-18 populations showed strong co-segregation of LLS-linked markers with the phenotype. From these genetic resources, six superior genotypes were identified. RIL 78-1 was resistant to LLS and rust, and recorded 30 % more pod yield than GPBD 4 (control). It also had higher kernel yield and oil yield along with higher oleate and linoleate content over GPBD 4. These genetic and genomic resources could be useful in breeding for LLS and rust resistance in peanut

The Role of Purported Mucoprotectants in Dealing with Irritable Bowel Syndrome, Functional Diarrhea, and Other Chronic Diarrheal Disorders in Adults

Chronic diarrhea is a frequent presenting symptom, both in primary care medicine and in specialized gastroenterology units. It is estimated that more than 5% of the global population suffers from chronic diarrhea. and that about 40% of these subjects are older than 60 years. The clinician is frequently faced with the need to decide which is the best therapeutic approach for these patients. While the origin of chronic diarrhea is diverse, impairment of intestinal barrier function, dysbiosis. and mucosal micro-inflammation are being increasingly recognized as underlying phenomena characterizing a variety of chronic diarrheal diseases. In addition to current pharmacological therapies, there is growing interest in alternative products such as mucoprotectants, which form a mucoadhesive film over the epithelium to reduce and protect against the development of altered intestinal permeability, dysbiosis, and mucosal micro-inflammation. This manuscript focuses on chronic diarrhea in adults, and we will review recent evidence on the ability of these natural compounds to improve symptoms associated with chronic diarrhea and to exert protective effects for the intestinal barrier