REPAIRtoire—a database of DNA repair pathways

REPAIRtoire is the first comprehensive database resource for systems biology of DNA damage and repair. The database collects and organizes the following types of information: (i) DNA damage linked to environmental mutagenic and cytotoxic agents, (ii) pathways comprising individual processes and enzymatic reactions involved in the removal of damage, (iii) proteins participating in DNA repair and (iv) diseases correlated with mutations in genes encoding DNA repair proteins. REPAIRtoire provides also links to publications and external databases. REPAIRtoire contains information about eight main DNA damage checkpoint, repair and tolerance pathways: DNA damage signaling, direct reversal repair, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination repair, nonhomologous end-joining and translesion synthesis. The pathway/protein dataset is currently limited to three model organisms: Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. The DNA repair and tolerance pathways are represented as graphs and in tabular form with descriptions of each repair step and corresponding proteins, and individual entries are cross-referenced to supporting literature and primary databases. REPAIRtoire can be queried by the name of pathway, protein, enzymatic complex, damage and disease. In addition, a tool for drawing custom DNA–protein complexes is available online. REPAIRtoire is freely available and can be accessed at http://repairtoire.genesilico.pl/

Deciphering the interstrand crosslink DNA repair network expressed by Trypanosoma brucei

Interstrand crosslinks (ICLs) represent a highly toxic form of DNA damage that can block essential biological processes including DNA replication and transcription. To combat their deleterious effects all eukaryotes have developed cell cycle-dependent repair strategies that co-opt various factors from ‘classical’ DNA repair pathways to resolve such lesions. Here, we report the first systematic dissection of how ICL repair might operate in the Trypanosoma brucei, the causative agent of African trypanosomiasis, and demonstrated that this diverged eukaryote expresses systems that show some intriguing differences to those mechanisms present in other organisms. Following the identification of trypanosomal homologues encoding for CSB, EXO1, SNM1, MRE11, RAD51 and BRCA2, gene deletion coupled with phenotypic studies demonstrated that all the above factors contribute to this pathogen’s ICL REPAIRtoire with their activities split across two epistatic groups. We postulate that one network, which encompasses TbCSB, TbEXO1 and TbSNM1, may operate throughout the cell cycle to repair ICLs encountered by transcriptional detection mechanisms while the other relies on homologous recombination enzymes (MRE11, RAD51 and BRCA2) that together help resolve lesions responsible for the stalling of DNA replication forks. This study not only sheds light on the conservation and divergence of ICL repair in one of only a handful of protists that can be studied genetically, but offers the promise of developing or exploiting ICL-causing agents as new anti-parasite therapies

Targeting DNA repair pathways for cancer treatment: what's new?

Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses

DNA Interstrand Crosslink Repair in Trypanosoma brucei

PhDGenomes are constantly challenged by agents that promote DNA damage, with interstrand crosslinks (ICLs) representing a particularly dangerous lesion. Ongoing work in the Wilkinson laboratory aimed at identifying novel agents that target Trypanosoma brucei, the causative agent of African trypanosomiasis, identified several prodrugs that once activated form ICLs in this protozoan parasite. To understand the complexity of ICL repair systems that T. brucei employs to resolve such damage, a variety of null mutant lines were generated that lack activities postulated to fix such lesions. Phenotypic screens using various DNA damaging agents revealed that TbMRE11, TbEXO1, TbCSB, TbCHL1, TbFAN1, TbBRCA2 and TbRAD51 all help to resolve ICLs, implicating components of the homologous recombination, nucleotide excision repair and mismatch repair pathways in resolving this form of damage: This approach demonstrated that components of the translesion synthesis pathway (TbREV2 and TbREV3) do not play a significant role in ICL repair. In many organisms, nucleases belonging to the SNM1/PSO2 family play a key and specific role in the repair of ICLs with this property extending to the T. brucei homologue, TbSNM1. To assess whether there is a functional linkage between the DNA repair factors noted above and TbSNM1, a series of double null mutants were constructed and the susceptibility of these lines to ICL inducing agents determined. Identification of their epistatic/non-epistatic interactions revealed that T. brucei expresses at least two ICL repair systems with one pathway involving the concerted activities of TbSNM1/TbCSB/TbEXO1, that we postulate functions to repair ICLs encountered by the transcriptional machinery, while the other is centred upon TbMRE11/TbFAN1/TbEXO1 that may help resolve lesions which cause stalling of DNA replication forks. By unravelling how T. brucei repairs ICLs, specific inhibitors against key components of these pathways could be developed and used in combination with DNA damaging agents to target trypanosomal infections.Queen Mary University of Londo

Exploring the Potential of Exome Sequencing in Idiopathic Azoospermia : A Genetic Burden and Network Analysis Study

Publisher Copyright: © 2023 Baiba Alkšere et al.The purpose of this study was to investigate the linkage of the association of azoospermia phenotype with genetic alterations, involved in genome instability. Male infertility is a multifactorial pathology, and genetic alterations might be the underlying factors in majority of cases of severe male infertility. The recent emergence of next-generation sequencing offers an opportunity to analyze many genes and their interactions at once, and whole-exome sequencing (WES) together with whole-genome sequencing (WGS) was recently suggested for implementation of diagnosis workup in severe infertility cases. However, the reports on WES in conjunction with burden tests and gene network analysis are scarce or lacking in cases of severe male infertility. WES was performed on 21 nonobstructive azoospermia patients. DNA samples were sequenced using the Twist Comprehensive Exome Panel. Genetic burden test was performed with Testing Rare vAriants using Public Data. Protein interactions were investigated with ConsensusPathDB and Cytoscape. For single nucleotide variants and copy number variations (CNV) analysis, samples were analyzed with the Illumina's BaseSpace Variant Interpreter. Genetic variant burden was found elevated in 1,473 genes out of 30,000 known testis expressed genes. Three hundred and two genes with increased loss-of-function (LoF) variant set were present in more than one sample. Overrepresentation analysis with pathway-based set of genes with high variant burden demonstrated 26 pathways. Overrepresentation analysis with protein complex-based gene sets obtained 14 sets, showing the involvement in cell proliferation and DNA repair. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) network analysis with Cytoscape identified two clusters: (1) genes, involved in DNA binding/condensation and repair processes and (2) genes with the role in ribosome biosynthesis and gene expression processes. Increased loss of function germline variant burden and sumoylation may have critical significance in spermatogenesis. These parameters may be used for focused diagnosis in nonobstructive azoospermia patients. This may have both general significance for the decreased organism functionality but in particular is critical in spermatogenesis.Peer reviewe

The 2011 Nucleic Acids Research Database Issue and the online Molecular Biology Database Collection

The current 18th Database Issue of Nucleic Acids Research features descriptions of 96 new and 83 updated online databases covering various areas of molecular biology. It includes two editorials, one that discusses COMBREX, a new exciting project aimed at figuring out the functions of the ‘conserved hypothetical’ proteins, and one concerning BioDBcore, a proposed description of the ‘minimal information about a biological database’. Papers from the members of the International Nucleotide Sequence Database collaboration (INSDC) describe each of the participating databases, DDBJ, ENA and GenBank, principles of data exchange within the collaboration, and the recently established Sequence Read Archive. A testament to the longevity of databases, this issue includes updates on the RNA modification database, Definition of Secondary Structure of Proteins (DSSP) and Homology-derived Secondary Structure of Proteins (HSSP) databases, which have not been featured here in >12 years. There is also a block of papers describing recent progress in protein structure databases, such as Protein DataBank (PDB), PDB in Europe (PDBe), CATH, SUPERFAMILY and others, as well as databases on protein structure modeling, protein–protein interactions and the organization of inter-protein contact sites. Other highlights include updates of the popular gene expression databases, GEO and ArrayExpress, several cancer gene databases and a detailed description of the UK PubMed Central project. The Nucleic Acids Research online Database Collection, available at: http://www.oxfordjournals.org/nar/database/a/, now lists 1330 carefully selected molecular biology databases. The full content of the Database Issue is freely available online at the Nucleic Acids Research web site (http://nar.oxfordjournals.org/)

Building a map of the breast cancer proteome - Strategies to increase coverage

Amongst the various –omics sciences, proteomics has the highest potential for functional characterization and consequently can contribute significantly to the field of cancer research. In particular, the focus of this thesis is on breast cancer. Alas, since state-of-the-art technologies cannot meet the complexity of upper eukaryotic proteomes, a complete resolution of clinical samples is still unachievable. Comprehensive mapping of proteins involved in cancer and of their PTMs is proposed in this thesis as a general strategy to increase the output of mass-spectrometry based proteomics. Different approaches to improve the coverage of this map are proposed: optimization of sample fractionation, focusing on difficult sub-proteomes, targeting of specific biological processes and optimization of data analysis. A combination of these approaches will provide a growing collection of empirical MS-spectra, which will enhance the detection by shotgun proteomics and facilitate the transition towards the development of targeted assays

Methods to increase the efficiency of precise CRISPR genome editing

Pluripotente Stammzellen haben das Potential, in unterschiedliche Zelltypen zu differenzieren und können genutzt werden, um organähnliche Mikrostrukturen zu generieren. Somit können molekulare Unterschiede verschiedenster künstlich differenzierter Gewebe, etwa zwischen Mensch und Schimpanse, anhand von pluripotenten Ausgangszellen untersucht werden. Da die Genome unserer nächsten ausgestorbenen Verwandten Neandertaler und Denisovaner aus konservierter DNA in alten Knochen sequenziert wurden, könnten ebenso Unterschiede zwischen Mensch und diesen Spezies oder dem letzten gemeinsamen Vorfahren untersucht werden. Dies erfordert jedoch die Generierung neandertalisierter Stammzellen durch künstliche Integration von Neandertalerallelen in humane Stammzellen, etwa durch die CRISPR Genomeditierungstechnik. Durch CRISPR kann ein DNA-Doppelstrangbruch an einer gewünschten Stelle im Genom eingefügt werden. Die zelluläre Reparatur des Doppelstrangbruchs ermöglicht dann die Editierung des Genoms. Basierend auf einer DNA-Matrize, die die gewünschte Modifikation trägt, kann das Genom an dieser Stelle präzise editiert werden. Die Effizienz präziser Editierung ist jedoch sehr niedrig im Vergleich zu unpräziser Reparatur. Um möglichst effizient neandertalisierte Stammzellen generieren zu können, wurden im Zuge dieser Doktorarbeit Methoden entwickelt, welche die präzise Genomeditierungseffizienz drastisch steigern. Zum einen wurde aus mehreren niedermolekularen Substanzen, welche mit Proteinen der DNA-Reparaturen interagieren, ein optimierter Mix entwickelt. Weiterhin konnte durch eine Mutation in einem zentralen Reparaturprotein die Effizienz für die Editierung eines einzelnen Gens auf 87% erhöht werden. Diese hohe Effizienz ermöglicht erstmals die präzise homozygote Editierung von vier Genen auf einmal in ein und derselben Zell