A Novel 3-Methyladenine DNA Glycosylase from Helicobacter pylori Defines a New Class within the Endonuclease III Family of Base Excision Repair Glycosylases

The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.Fil: O'Rourke, Eyleen J.. Centre National de la Recherche Scientifique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Chevalier, Catherine. Instituto Pasteur; FranciaFil: Boiteux, Serge. Centre National de la Recherche Scientifique; FranciaFil: Labigne, Agnès. Instituto Pasteur; FranciaFil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Radicella, Juan Pablo. Centre National de la Recherche Scientifique; Franci

An image analysis toolbox for high-throughput C. elegans assays

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.National Institutes of Health (U.S.) (U54 EB005149

Identification, characterization and biological relevance of base excision repair DNA glycosylases from Helicobacter pylori

Fil: O'Rourke, Eyleen J.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Helicobacter pylori Genes Involved in Avoidance of Mutations Induced by 8-Oxoguanine

International audienceABSTRACT Chromosomal rearrangements and base substitutions contribute to the large intraspecies genetic diversity of Helicobacter pylori . Here we explored the base excision repair pathway for the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), a ubiquitous form of oxidized guanine. In most organisms, 8-oxoG is removed by a specific DNA glycosylase (Fpg in bacteria or OGG1 in eukaryotes). In the case where replication of the lesion yields an A/8-oxoG base pair, a second DNA glycosylase (MutY) can excise the adenine and thus avoid the fixation of the mutation in the next round of replication. In a genetic screen for H. pylori genes complementing the hypermutator phenotype of an Escherichia coli fpg mutY strain, open reading frame HP0142, a putative MutY coding gene, was isolated. Besides its capacity to complement E. coli mutY strains, HP0142 expression resulted in a strong adenine DNA glycosylase activity in E. coli mutY extracts. Consistently, the purified protein also exhibited such an activity. Inactivation of HP0142 in H. pylori resulted in an increase in spontaneous mutation frequencies. An Mg-dependent AP (abasic site) endonuclease activity, potentially allowing the processing of the abasic site resulting from H. pylori MutY activity, was detected in H. pylori cell extracts. Disruption of HP1526, a putative xth homolog, confirmed that this gene is responsible for the AP endonuclease activity. The lack of evidence for an Fpg/OGG1 functional homolog is also discussed

Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases

International audienc

Genetic variability and DNA repair: base excision repair activities in Helicobacter pylori .

One of the remarkable characteristics of Helicobacter pylori is the high genetic diversity it displays. Based on the genome sequencing results, the absence of certain DNA repair activities has been postulated to be one of the causes for the genetic variability of this pathogen. We explored the possible base excision repair (BER) pathways present in H. pylori. We analyzed the activities corresponding to the enzymes participating in the first two steps of the pathway, the DNA glycosylases, specific for each kind of base damage, and the endonuclease that cleaves the resulting abasic (AP) site. We review here the data on the repair of alkylating DNA damage and oxidized pyrimidines and present results on studies carried out on bacterial extracts and purified proteins for the other BER activities. The combined approaches allowed the identification of a 3-methyl adenine DNA glycosylase, an endonuclease III, a uracil glycosylase, an adenine DNA glycosylase specific for 8-oxoguanine/adenine base pairs, and an AP endonuclease activity. We also discuss the possible role of the host in the bacterial genetic variability and the potential appearance of new alleles that could influence H. pylori persistence.Fil: O'Rourke, Eyleen J.. Centre National de la Recherche Scientifique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mathieu, Aurelie. Centre National de la Recherche Scientifique; FranciaFil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Radicella, Juan Pablo. Centre National de la Recherche Scientifique; Franci

What are housekeeping genes?

The concept of "housekeeping gene" has been used for four decades but remains loosely defined. Housekeeping genes are commonly described as "essential for cellular existence regardless of their specific function in the tissue or organism", and "stably expressed irrespective of tissue type, developmental stage, cell cycle state, or external signal". However, experimental support for the tenet that gene essentiality is linked to stable expression across cell types, conditions, and organisms has been limited. Here we use genome-scale functional genomic screens together with bulk and single-cell sequencing technologies to test this link and optimize a quantitative and experimentally validated definition of housekeeping gene. Using the optimized definition, we identify, characterize, and provide as resources, housekeeping gene lists extracted from several human datasets, and 10 other animal species that include primates, chicken, and C. elegans. We find that stably expressed genes are not necessarily essential, and that the individual genes that are essential and stably expressed can considerably differ across organisms; yet the pathways enriched among these genes are conserved. Further, the level of conservation of housekeeping genes across the analyzed organisms captures their taxonomic groups, showing evolutionary relevance for our definition. Therefore, we present a quantitative and experimentally supported definition of housekeeping genes that can contribute to better understanding of their unique biological and evolutionary characteristics

Evidence for the Active Role of a Novel Nuclease from Helicobacter pylori in the Horizontal Transfer of Genetic Information

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems

StanDep: Capturing transcriptomic variability improves context-specific metabolic models.

Diverse algorithms can integrate transcriptomics with genome-scale metabolic models (GEMs) to build context-specific metabolic models. These algorithms require identification of a list of high confidence (core) reactions from transcriptomics, but parameters related to identification of core reactions, such as thresholding of expression profiles, can significantly change model content. Importantly, current thresholding approaches are burdened with setting singular arbitrary thresholds for all genes; thus, resulting in removal of enzymes needed in small amounts and even many housekeeping genes. Here, we describe StanDep, a novel heuristic method for using transcriptomics to identify core reactions prior to building context-specific metabolic models. StanDep clusters gene expression data based on their expression pattern across different contexts and determines thresholds for each cluster using data-dependent statistics, specifically standard deviation and mean. To demonstrate the use of StanDep, we built hundreds of models for the NCI-60 cancer cell lines. These models successfully increased the inclusion of housekeeping reactions, which are often lost in models built using standard thresholding approaches. Further, StanDep also provided a transcriptomic explanation for inclusion of lowly expressed reactions that were otherwise only supported by model extraction methods. Our study also provides novel insights into how cells may deal with context-specific and ubiquitous functions. StanDep, as a MATLAB toolbox, is available at https://github.com/LewisLabUCSD/StanDep