1,720,994 research outputs found
Screening of pipolins in Bacteria and detection of genetic exchanges with other MGEs
No full textThe authors have carried out a massive screening of pipolins in the bacterial genome database "Assembly" (NCBI). Then, they found integrases and new integration sites associated to pipolins. Pipolins and other MGEs (plasmids, ciMGEs, and phages) were annotated to characterize pipolin genetic content comparatively. Finally, they applied a recent method to detect recent genetic exchange events between pipolins and other MGEs based on the weighted gene repertoire relatedness and protein sequence identity.
This dataset contains several files created throughout the analysis. Files 1 and 2 are related to pipolin screening and plotting. Files 3 to 5 contain the list of genes classified as "recombining" or "non-recombining" encoded in the MGEs.</p
Comparison of MDA methods
No full textComparison of whole metagenome amplification competence by new piPolB-based MDA methods and commercial kits using a mini mock-metagenome containing high-GC sequences.
Mini mock metagenome made up of E. coli, B. subtilis, P. aeruginosa and K. rhizophila genomes. A total of 11 samples were analyzed:
unamplified metagenome (NA and NA2)
samples obtained by random-primers MDA (RP-MDA and RP-MDA2)
PrimPol-MDA
new MDA with piPolB as only enzyme
piPolB with a previous DNA alkaline denaturation step (piPolB+D)
new MDA with piPolB and phi29 DNA polymerases (piMDA and piMDA2)
and the latter method with a previous denaturation step (piMDA+D and piMDA+D2)</ul
Thermus thermophilus TnSeq
No full textIn this study we develop a procedure for thermostable selection of random insertion mutants based on a gene cassette encoding a thermostable resistance to kanamycin flanked by the recognition sites (ME sites) for Tn5 transposase. The transposition library generated in vitro was initialy transformed in a PrimPol mutant HB27 derivative (ppol), which shows ~2 log fold transformation efficiency. This allowed the generation of the generation of a large library of insertion mutants (“Mother”), which was later transferred to a wild type HB27 strain (“Daughter”). Both libraries were sequenced straightforward and after several culture passages. See the manuscript for more details.
This dataset contains the raw data of the sequencing libraries as well as the GitHub repository containing the scrips and reports from the analysis.</p
ADN polimerasas independientes de cebador y su uso para la síntesis de ADN
No full textLa presente invención proporciona un péptido aislado de SEQ ID NO: 1 necesario para las enzimas primasa activas así como nuevas enzimas ADN polimerasa replicativas, preferiblemente la de SEQ ID NO: 2, que comprende dicho péptido. Por tanto, estas ADN polimerasas están dotadas de actividad de cebado y no requieren cebadores proporcionados externamente para iniciar y realizar la amplificación del ADN. Estas polimerasas son capaces de llevar a cabo una síntesis de ADN de novo fiel y procesiva de plantillas de ADN en ausencia de cebadores presintetizados. Por lo tanto, estas enzimas de la invención actúan como primasas y ADN polimerasas. Además, muestran capacidad de síntesis de translesiones, por lo que pueden ser útiles no sólo para la amplificación de todo el genoma sino también para la amplificación de ADN dañado. La invención se refiere además a métodos para amplificar moldes de ADN usando estas enzimas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Institut PasteurT3 Traducción de patente europe
Primerunabhängige DNA-polymerasen und ihre verwendung zur DNA-synthese
No full textThe present invention provides an isolated peptide of SEQ ID NO: 1 needed for primase active as well as new replicative DNA polymerase enzymes, preferably that of SEQ ID NO: 2, comprising said peptide. Thus, these DNA polymerases are endowed with priming activity and do not require externally provided primers for initiating and performing DNA amplification. These polymerases are able to carry out a faithful and processive de novo DNA synthesis of DNA templates in the absence of pre-synthetized primers. Therefore, these enzymes of the invention act both as primases and DNA polymerases. Furthermore, they show translesion synthesis capacity, so that they may be useful not only for whole-genome amplification but also for the amplification of damaged DNAs. The invention further refers to methods for amplifying templates DNAs using these enzymes.Peer reviewedConsejo Superior de Investigaciones Científicas (CSIC), Institut PasteurB1 Patente sin examen previ
Primer-independent DNA polymerases and their use for DNA synthesis
No full textThe present invention provides an isolated peptide of SEQ ID NO: 1 needed for primase active as well as new replicative DNA polymerase enzymes, preferably that of SEQ ID NO: 2, comprising said peptide. Thus, these DNA polymerases are endowed with priming activity and do not require externally provided primers for initiating and performing DNA amplification. These polymerases are able to carry out a faithful and processive de novo DNA synthesis of DNA templates in the absence of pre-synthetized primers. Therefore, these enzymes of the invention act both as primases and DNA polymerases. Furthermore, they show translesion synthesis capacity, so that they may be useful not only for whole-genome amplification but also for the amplification of damaged DNAs. The invention further refers to methods for amplifying templates DNAs using these enzymesPeer reviewedConsejo Superior de Investigaciones Científicas (España), Institut PasteurA1 Solicitud de patente con informe sobre el estado de la técnic
La endonucleasa AP del virus de la peste porcina y el papel biológico del sistema viral de reparación del ADN
No full textTesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-02-2009African swine fever virus (ASFV) is a deoxyvirus that produces a lethal disease in domestic pigs. The ASFV genome encodes several proteins that might constitute a viral base excision repair system (BER), possibly aimed to the maintenance of viral genome stability. In the infected swine the virus mainly replicates in monocytes and macrophages, which may produce reactive oxygen species (ROS) as a response to the infection, thus threatening the integrity of the viral DNA. The viral DNA repair system includes an EndoIV-like AP endonuclease (named pE296R), a DNA repair polymerase (pol X), a PCNA-like protein (pE301R) and a DNA ligase. The biochemical properties of ASFV pol X have been studied before. In this work, we have characterized the biochemical properties of the recombinant purified viral AP endonuclease. The structural properties analyzed and DNA repair enzymatic proper-ties of protein pE296R —AP endonucleolytic, 3’5’ exonuclease, 3’-diesterase and nucleotide inci-sion repair (NIR) activities— are similar to those previously shown for other EndoIV-like AP endonu-cleases. The characterization of the 3’5’ exonuclease and 3’-repair diesterase of pE296R suggests that it has a strong preference for mispaired and oxidative base lesions at the 3’-termini of single-strand breaks. Furthermore, the expression of E296R gene in a E. coli strain lacking AP endonucle-ases promotes cell survival under chronic treatment with alkylating and oxidative compounds, indi-cating a repair activity in vivo of different DNA damages. We also found that all the enzyme repair activities are inhibited by reducing agents. Binding of the viral AP endonuclease to DNA is suppressed by reducing agents, which might account for the in-hibition of the enzyme’s activities. Moreover, the pE296R protein contains intramolecular disulfide bonds. Therefore, formation and breakage of the disulfide bond may provide a regulatory mecha-nism of the enzyme’s DNA repair activities. Finally, we have also tried to address the biological role of the viral DNA repair system. First, we analyzed the accumulation and intracellular distribution of the viral proteins pE301R, pE296R and pol X. The pattern of distribution of these proteins in the context of viral infection is compatible with shared a role together in DNA repair, but also with other possible unique functions for each protein. Using ASFV deletion mutants in genes E296R and pol X, we have determined that the viral endonuc-lease is essential for virus growth in macrophages, but not in Vero cells. The viral pol X is also re-quired in macrophages but only under conditions of repetitive infection cycles. The biochemical and genetic properties of ASFV AP endonuclease and pol X are consistent with the repair of DNA damage generated by the genotoxic intracellular environment of the host macrophage. This work supports the existence of a viral reparative system to maintain virus viability in the infected macrophage
Multiple roles of genome-attached bacteriophage terminal proteins
No full textAbstractProtein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer
DNA polymerases for whole genome amplification: considerations and future directions
Full text linkIn the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directionsThis research was funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe, grant PID2021-123403NB-I00 to M.R.-
Pipolins are bimodular platforms that maintain a reservoir of defense systems exchangeable with various bacterial genetic mobile elements
No full textThe dataset that supports the findings of this study are archived in the Universidad Autónoma de Madrid data repository e‐cienciaDatos in https://doi.org/10.21950/QG3QEEDefense genes gather in diverse types of genomic islands in bacteria and provide immunity against viruses and other genetic mobile elements. Here, we disclose pipolins, previously found in diverse bacterial phyla and encoding a primer-independent PolB, as a new category of widespread defense islands. The analysis of the occurrence and structure of pipolins revealed that they are commonly integrative elements flanked by direct repeats in Gammaproteobacteria genomes, mainly Escherichia, Vibrio or Aeromonas, often taking up known mobile elements integration hotspots. Remarkably, integrase dynamics correlates with alternative integration spots and enables diverse lifestyles, from integrative to mobilizable and plasmid pipolins, such as in members of the genera Limosilactobacillus, Pseudosulfitobacter or Staphylococcus. Pipolins harbor a minimal core and a large cargo module enriched for defense factors. In addition, analysis of the weighted gene repertoire relatedness revealed that many of these defense factors are actively exchanged with other mobile elements. These findings indicate pipolins and, potentially other defense islands, act as orthogonal reservoirs of defense genes, potentially transferable to immune autonomous MGEs, suggesting complementary exchange mechanisms for defense genes in bacterial populationsThis work was funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe [PID2021-123403NB-I00] and Comunidad de Madrid (V PRICIT call Research Grants for Young Researchers from Universidad Autónoma de Madrid) [SI3/PJI/2021–00271]. VMC was holder of an FPI-UAM PhD Fellowship from UAM [SFPI/2023–00603]. Funding for open access charge: AEI [PID2021-123403NB-I00]
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