Updates in Rhea-a manually curated resource of biochemical reactions.

Rhea (http://www.ebi.ac.uk/rhea) is a comprehensive and non-redundant resource of expert-curated biochemical reactions described using species from the ChEBI (Chemical Entities of Biological Interest) ontology of small molecules. Rhea has been designed for the functional annotation of enzymes and the description of genome-scale metabolic networks, providing stoichiometrically balanced enzyme-catalyzed reactions (covering the IUBMB Enzyme Nomenclature list and additional reactions), transport reactions and spontaneously occurring reactions. Rhea reactions are extensively curated with links to source literature and are mapped to other publicly available enzyme and pathway databases such as Reactome, BioCyc, KEGG and UniPathway, through manual curation and computational methods. Here we describe developments in Rhea since our last report in the 2012 database issue of Nucleic Acids Research. These include significant growth in the number of Rhea reactions and the inclusion of reactions involving complex macromolecules such as proteins, nucleic acids and other polymers that lie outside the scope of ChEBI. Together these developments will significantly increase the utility of Rhea as a tool for the description, analysis and reconciliation of genome-scale metabolic models

Updates in Rhea - an expert curated resource of biochemical reactions.

Rhea (http://www.rhea-db.org) is a comprehensive and non-redundant resource of expert-curated biochemical reactions designed for the functional annotation of enzymes and the description of metabolic networks. Rhea describes enzyme-catalyzed reactions covering the IUBMB Enzyme Nomenclature list as well as additional reactions, including spontaneously occurring reactions, using entities from the ChEBI (Chemical Entities of Biological Interest) ontology of small molecules. Here we describe developments in Rhea since our last report in the database issue of Nucleic Acids Research. These include the first implementation of a simple hierarchical classification of reactions, improved coverage of the IUBMB Enzyme Nomenclature list and additional reactions through continuing expert curation, and the development of a new website to serve this improved dataset

Updates in Rhea: SPARQLing biochemical reaction data.

Rhea (http://www.rhea-db.org) is a comprehensive and non-redundant resource of over 11 000 expert-curated biochemical reactions that uses chemical entities from the ChEBI ontology to represent reaction participants. Originally designed as an annotation vocabulary for the UniProt Knowledgebase (UniProtKB), Rhea also provides reaction data for a range of other core knowledgebases and data repositories including ChEBI and MetaboLights. Here we describe recent developments in Rhea, focusing on a new resource description framework representation of Rhea reaction data and an SPARQL endpoint (https://sparql.rhea-db.org/sparql) that provides access to it. We demonstrate how federated queries that combine the Rhea SPARQL endpoint and other SPARQL endpoints such as that of UniProt can provide improved metabolite annotation and support integrative analyses that link the metabolome through the proteome to the transcriptome and genome. These developments will significantly boost the utility of Rhea as a means to link chemistry and biology for a more holistic understanding of biological systems and their function in health and disease

Positive Regulation of DNA Double Strand Break Repair Activity during Differentiation of Long Life Span Cells: The Example of Adipogenesis

Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs), and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE) and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ) that relies on the DNA dependent protein kinase (DNA-PK) activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue

Age-Related Neuronal Degeneration: Complementary Roles of Nucleotide Excision Repair and Transcription-Coupled Repair in Preventing Neuropathology

Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR–deficient Csa−/− and Csb−/− CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER–deficient Xpa−/− and Xpc−/− XP mice, but also occurred in XpdXPCS mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR–deficient mice are compatible with focal dysmyelination in CS patients. Both TCR–deficient and NER–deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa−/−, Csb−/−) or highly sporadic (Xpa−/−, Xpc−/−) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR–deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa−/− and Csb−/− TCR–deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR–deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival

Involvement of global genome repair, transcription coupled repair, and chromatin remodeling in UV DNA damage response changes during development

Nucleotide Excision Repair (NER), which removes a variety of helix-distorting lesions from DNA, is initiated by two distinct DNA damage-sensing mechanisms. Transcription Coupled Repair (TCR) removes damage from the active strand of transcribed genes and depends on the SWI/SNF family protein CSB. Global Genome Repair (GGR) removes damage present elsewhere in the genome and depends on damage recognition by the XPC/RAD23/Centrin2 complex. Currently, it is not well understood to what extent both pathways contribute to genome maintenance and cell survival in a developing organism exposed to UV light. Here, we show that eukaryotic NER, initiated by two distinct subpathways, is well conserved in the nematode Caenorhabditis elegans. In C. elegans, involvement of TCR and GGR in the UV-induced DNA damage response changes during development. In germ cells and early embryos, we find that GGR is the major pathway contributing to normal development and survival after UV irradiation, whereas in later developmental stages TCR is predominantly engaged. Furthermore, we identify four ISWI/Cohesin and four SWI/SNF family chromatin remodeling factors that are implicated in the UV damage response in a developmental stage dependent manner. These in vivo studies strongly suggest that involvement of different repair pathways and chromatin remodeling proteins in UV-induced DNA repair depends on developmental stage of cells

Ly49H signaling through DAP10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection

The activating natural killer (NK) cell receptor Ly49H recognizes the mouse cytomegalovirus (MCMV) m157 glycoprotein expressed on the surface of infected cells and is required for protection against MCMV. Although Ly49H has previously been shown to signal via DAP12, we now show that Ly49H must also associate with and signal via DAP10 for optimal function. In the absence of DAP12, DAP10 enables Ly49H-mediated killing of m157-bearing target cells, proliferation in response to MCMV infection, and partial protection against MCMV. DAP10-deficient Ly49H+ NK cells, expressing only Ly49H–DAP12 receptor complexes, are partially impaired in their ability to proliferate during MCMV infection, display diminished ERK1/2 activation, produce less IFN-γ upon Ly49H engagement, and demonstrate reduced control of MCMV infection. Deletion of both DAP10 and DAP12 completely abrogates Ly49H surface expression and control of MCMV infection. Thus, optimal NK cell–mediated immunity to MCMV depends on Ly49H signaling through both DAP10 and DAP12

Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV–induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPFR153P) were compared to an XP–causing mutation (XPFR799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPFR153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPFR153P-ERCC1 into the nucleus of XPF–deficient human cells restored nucleotide excision repair of UV–induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1

Transcriptional Mutagenesis Induced by 8-Oxoguanine in Mammalian Cells

Most of the somatic cells of adult metazoans, including mammals, do not undergo continuous cycles of replication. Instead, they are quiescent and devote most of their metabolic activity to gene expression. The mutagenic consequences of exposure to DNA–damaging agents are well documented, but less is known about the impact of DNA lesions on transcription. To investigate this impact, we developed a luciferase-based expression system. This system consists of two types of construct composed of a DNA template containing an 8-oxoguanine, paired either with a thymine or a cytosine, placed at defined positions along the transcribed strand of the reporter gene. Analyses of luciferase gene expression from the two types of construct showed that efficient but error-prone transcriptional bypass of 8-oxoguanine occurred in vivo, and that this lesion was not repaired by the transcription-coupled repair machinery in mammalian cells. The analysis of luciferase activity expressed from 8OG:T-containing constructs indicated that the magnitude of erroneous transcription events involving 8-oxoguanine depended on the sequence contexts surrounding the lesion. Additionally, sequencing of the transcript population expressed from these constructs showed that RNA polymerase II mostly inserted an adenine opposite to 8-oxoguanine. Analysis of luciferase expression from 8OG:C-containing constructs showed that the generated aberrant mRNAs led to the production of mutant proteins with the potential to induce a long-term phenotypical change. These findings reveal that erroneous transcription over DNA lesions may induce phenotypical changes with the potential to alter the fate of non-replicating cells

Acute myeloid leukemia of donor origin after allogeneic stem cell transplantation from a sibling who harbors germline XPD and XRCC3 homozygous polymorphisms

A 54-year-old woman was diagnosed with infiltrative ductal breast carcinoma. Two years after treatment, the patient developed an acute myeloid leukemia (AML) which harbored del(11q23) in 8% of the blast cells. The patient was submitted for allogeneic stem cell transplantation (aSCT) from her HLA-compatible sister. Ten months after transplantation, she relapsed with an AML with basophilic maturation characterized by CD45low CD33high, CD117+, CD13-/+, HLA Drhigh, CD123high, and CD203c+ blast cells lacking expression of CD7, CD10, CD34, CD15, CD14, CD56, CD36, CD64, and cytoplasmic tryptase. Karyotype analysis showed the emergence of a new clone with t(2;14) and FISH analysis indicated the presence of MLL gene rearrangement consistent with del(11q23). Interestingly, AML blast cell DNA tested with microsatellite markers showed the same pattern as the donor's, suggesting that this AML emerged from donor cells. Additionally, polymorphisms of the XPA, XPD, XRCC1, XRCC3 and RAD51 DNA repair genes revealed three unfavorable alleles with low DNA repair capacity