Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.)

A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%–3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about I%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels

The human DNA glycosylases NEIL1 and NEIL3 excise psoralen-induced DNA-DNA cross-links in a four-stranded DNA structure

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks

Served Through Service: Undergraduate Students’ Experiences in Community Engaged Learning at a Catholic and Marianist University

Students participating in sustained community service at an urban Catholic and Marianist university were volunteer informants in this qualitative exploration of the meaning they make of their service experiences. A PhD student research team (nine members) interviewed fourteen undergraduate students (eleven of whom were seniors). Findings were organized as themes constructed within three domains: background, experience, and meaning. Within “background,” students who had prior work in faith-based service before college deepened their meaning of service. Within “experience,” there were social and cultural dynamics of navigating on and off campus life, including the roles students played as well as the challenge of time management. Within “meaning,” building relationships was central to community service. Students built strong personal relationships with and deep commitments to city residents; the meaning of their own identities grew and developed. Experiencing the roots of social injustice led students both to confirm and to reconsider their life vocations

Served Through Service: Undergraduate Students’ Experiences in Community Engaged Learning at a Catholic and Marianist University

Students participating in sustained community service at an urban Catholic and Marianist university were volunteer informants in this qualitative exploration of the meaning they make of their service experiences. A PhD student research team (nine members) interviewed fourteen undergraduate students (eleven of whom were seniors). Findings were organized as themes constructed within three domains: background, experience, and meaning. Within “background,” students who had prior work in faith-based service before college deepened their meaning of service. Within “experience,” there were social and cultural dynamics of navigating on and off campus life, including the roles students played as well as the challenge of time management. Within “meaning,” building relationships was central to community service. Students built strong personal relationships with and deep commitments to city residents; the meaning of their own identities grew and developed. Experiencing the roots of social injustice led students both to confirm and to reconsider their life vocations. Estudiantes que participaban en servicios continuados a la comunidad en una universidad católica marianista urbana participaron como encuestados voluntarios de esta exploración cualitativa del significado que le dan a sus experiencias de servicio. Un equipo de investigación de estudiantes (nueve) de doctorado entrevistaron a catorce estudiantes de licenciatura (de los cuales 10 eran de último curso). Los resultados se organizaron en temas englobados en tres dominios: antecedentes, experiencia y significado. En “antecedentes”, los estudiantes que habían realizado trabajo previo en servicios religiosos antes de la universidad profundizaron su significado de servicio. Dentro de “experiencia”, hubo dinámicas sociales y culturales que navegaban dentro y fuera de la vida en el campus, desde el papel de los estudiantes hasta el reto de la gestión del tiempo. En “significado”, la construcción de relaciones resultó central para los servicios a la comunidad. Los estudiantes cultivaron fuertes relaciones personales y compromisos profundos con los residentes de la ciudad. El significado de su propia identidad creció y se desarrolló. Experimentar las raíces de la injusticia social orientó a los estudiantes a confirmar y reconsiderar su vocación y elección de vida. Palabras clave: servicio, aprendizaje a través del servicio, aprendizaje-servicio, aprendizaje comprometido con la comunidad, liderazgo estudiantil, estudiante de licenciatur

Regulation of the vapBC-1 Toxin-Antitoxin Locus in Nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) are human-adapted commensal bacteria that can cause a number of chronic mucosal infections, including otitis media and bronchitis. One way for these organisms to survive antibiotic therapy and cause recurrent disease is to stop replicating, as most antimicrobials target essential biosynthetic pathways. Toxin-antitoxin (TA) gene pairs have been shown to facilitate entry into a reversible bacteriostatic state. Characteristically, these operons encode a protein toxin and an antitoxin that associate following translation to form a nontoxic complex, which then binds to and regulates the cognate TA promoter. Under stressful conditions, the labile antitoxin is degraded and the complex disintegrates, freeing the stable toxin to facilitate growth arrest. How these events affected the regulation of the TA locus, as well as how the transcription of the operon was subsequently returned to its normal state upon resumption of growth, was not fully understood. Here we show that expression of the NTHi vapBC-1 TA locus is repressed by a complex of VapB-1 and VapC-1 under conditions favorable for growth, and activated by the global transactivator Factor for Inversion Stimulation (Fis) upon nutrient upshift from stationary phase. Further, we demonstrate for the first time that the VapC-1 toxin alone can bind to its cognate TA locus control region and that the presence of VapB-1 directs the binding of the VapBC-1 complex in the transcriptional regulation of vapBC-1