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    "title": "R&R lab @UAM",
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    "text": "21st August 2023 - New Paper Alert!\n\n\n\nIt took some time, but our new method for whole genome and metagenomes DNA amplification is just published in NAR Genomics and Bioinformatics: https://academic.oup.com/nargab/article/5/3/lqad073/7246555\nIn this work, we used the piPolB to develop new methods for WGA. We used the piPolB alone and in combination with a Φ29 DNA polymerase, a highly processive DNA polymerase. Remarkably, the latter proved to be a great protocol, outperforming all previously available methods In particular, our piMDA method provides very good and unbiased coverage for sequences with high GC content, usually the Achilles heel of most MDA methods.\nThis work was led by @CarlosDOC_, with the participation of @karmayoral from the lab. We were also very fortunate to be able to count on the collaboration of Dr. Conceiçao Egas from Biocant-CNC (Portugal).\n\n\n\nWelcome…\n\n\n\n… to the R’n’R lab, where you can learn about DNA Replication and Repair, meet Modesto Redrejo Rodríguez and listen to Rock & Roll music.\nOur lab is at the School of Medicine of UAM (Madrid) and it also belongs to the Instituto de Investigaciones Biomédicas “Alberto Sols”, a joint CSIC/UAM Research Institute on biomedicine. The RnR group is also available at IIB site.\n\n\nLab Scope\n\n\n\nIn our lab, we focus on the protein factors (enzymes) that carry out DNA replication and repair.\nDNA polymerases are the main responsible for copying the information stored in each DNA strand. We are also interested in other enzymes, such as DNA binding proteins, or DNA Glycosylases, and AP endonuclease, which can surgically identify and remove damage from the double helix. We carry out structure-function studies along with phylogenetic approaches to understand the mechanisms of DNA maintenance and the evolution of those mechanisms.\nWe use simple genetic models, like transposons, virus and bacteria to analyze the molecular mechanisms that maintain genetic information in the DNA molecule. This also allows us to devise new methods and biotechnological applications.\nWe are currently focused in DNA polymerases but new projects are being cooked under low heat!!\n\n\nPhylosophy\nWe are a small lab that aspires to be a good place to learn and science together. As the great Professor Margarita Salas used to say, our ultimate goal is “to work hard and to work well”. To achieve that, we team up in a collaborative science environment.\nWe use Benchling as electronic laboratory notebook for daily work. All new students get the following Benchling notes about the lab work:\n\nWorking at R’n’R lab\nTips for scientific figures and presentations\nGeneral tips for writing scientific texts\n\n\n\n\n\n\n\nDisclaimer\n\n\n\nThis is a Quarto website, hosted is on a GitHub repo under CC BY-NC license. To learn more about Quarto see https://quarto.org. You can also check our R (& Rstudio)course in the Teaching section.\nAlso, it is likely that you find some mistypes or even some big mistakes throughout these site. I will appreciate it if you let me know about anything that could be corrected or just improved."
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    "text": "About this site\n\n1 + 1\n\n[1] 2"
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    "text": "Current Members\n\nModesto Redrejo Rodríguez (PI, Assistant Professor)     \nModesto is Assistant Professor (in Spanish “Profesor Ayudante Doctor”) at the Biochemistry Department of the School of Medicine of one of the largest universities in Madrid, the Madrid Autonomous University (UAM).\nSince his PhD thesis, working on DNA repair mechanisms of ASFV (large DNA virus), he has been very interested in DNA replication and repair mechanisms and how these mechanisms evolved. After that, he moved to the Murat Saparbaev’s lab at the Gustave Roussy Institute (Villejuif, nearby Paris) to work on Base Excision Repair enzymes. Then, in 2011, he moved back to Madrid as Postdoc and later on Senior Research Associate in the Margarita Salas’ lab at the Molecular Biology Center (CBM).\nHe is very interested in the use of multidisciplinary approaches, using biochemistry, structural biology, genetics and bioinformatics to deal with biological problems. A full list of Modesto’s publications can be found here.\n\n\nJuan J. Arredondo Lamas (Associate Professor)\nJuan is a very experienced researcher in genetics and molecular biology of model organisms, particularly Drosophila melanogaster. After several months of sharing the lab with us and providing very interesting “outsider” discussions, he decided to join the crew. He will be very helpful in the students’ supervision and focus in the biological role of pipolins.\n\n\nEsmeralda Solar Venero (Postdoc, 2022-)\nEsme is an expert molecular microbiologist from Buenos Aires. She is highly interested in pipolins diversity and mobilization.\n\n\nCarmen Mayoral Campos (PhD Student, 2019- )\nCarmen is graduated in Biology and MSc in Biomolecules and Cell Dynamics (UAM). She is working in structure-function studies of piPolB, focused on understanding DNA priming capacity of these new DNA polymerases.\n\n\nVíctor Mateo Cáceres (PhD student, 2019-)\nVíctor started his BSc thesis working on the biological role of piPolBs in bacteria. His project involved the characterization of a piPolB mutant in the context of a naturally occurring pipolin from E. coli. However, during the Covid19 lockdown, he decided to switch into a Bioinformatics project and then enrolled in the Bioinformatics and Computational Biology. During the first year of his Master he got a “Collaboration Fellowship” that will help us to finish the ExplorePipolin pipeline.\n\n\nDiego Duarte Zara (Master Student, 2021-)\nDiego joined the lab in 2021 to work on piPolB biological role for his Bachelor’s thesis, working with Juan and Carmen. He obtained very interesting results that we will further explore during his Master’s thesis during 2023.\n\n\nNada Znaidi (Visiting PhD student, 2022-)\nNada is a PhD student from Université Tunis El Manar, directed by Prof. Samia Réjiba. Since September 2022, she spends some months ever year in our lab, analyzing the prevalence and genetic structure of pipolins from E. coli clinical isolates.\n\n\n\nLab pictures\n\n\n\nDecember 2023\n\n\n\n\n\nR’n’R Lab oficial Picture in Marzo 2022\n\n\n\n\n\n\n\nThe lab in September 2021\n\n\n\n\nAlumni\n\nJuan Carlos Martín Esteban (MSc Student, 2020-2022)\n\n\nAlejandro Serrano Sánchez (BSc Thesis, 2022)\n\n\nCarlos D. Ordóñez Cencerrado (PhD Student, 2017-2022)\nCarlos earned an MSc in Virology and he is interested in molecular biology, virology, biochemistry, structural biology and biotechnological application of virus. He carried out his PhD thesis working on the study of Translesion Synthesis capacity of B-family DNA polymerases also can carry out a faithful and processive DNA replication. He also performing structural-function studies and analysis of the influence of metal cofactor during the process.\nFind him on Researchgate,  @CarlosDOC_ or by e-mail: cordonez@cbm.csic.es\n\n\nMario Rodríguez Mestre (PhD Student, 2020-2021 )\n\n\n​Liubov Chuprikova (Bioinformatics MSc, 2019-2020)\nLiuba developed ExplorePipolin, a pipeline for the annotation and characterization of pipolins. Her Master thesis was carried out under the joint supervision of Modesto and María de Toro, head of the Genomics and Bioinformatics facility of CIBIR, and an expert in the annotation of prokaryotic genomes and mobilome. We are grateful to Liuba’s help after her Thesis, which allowed us to publish ExplorePipolin in 2022.\n\n\nIrene Díaz García (BSc Student, 2020-2021)\n\n\nLorenzo Vargas Román (BSc Student, 2020-2021)\n\n\nAna Lechuga Mateo (MSc and PhD Student, 2016-2020)\nAna carried out her MSc in Virology in Salas’ lab under Modesto’s supervision and then she decided to stay in the lab for Ph.D. She worked in two different but complementary aspects of Bam35 tectivirus biology. First, she is characterizing viral DNA binding proteins and their role in TP-DNA genome replication. Moreover, she has been the main responsible for the VirHost-omics project, using a Y2H screen and next-generation sequence to study virus-host relationships by high-through"
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    "text": "Alumni\n\nJuan Carlos Martín Esteban (MSc Student, 2020-2022)\n\n\nAlejandro Serrano Sánchez (BSc Thesis, 2022)\n\n\nCarlos D. Ordóñez Cencerrado (PhD Student, 2017-2022)\nCarlos earned an MSc in Virology and he is interested in molecular biology, virology, biochemistry, structural biology and biotechnological application of virus. He carried out his PhD thesis working on the study of Translesion Synthesis capacity of B-family DNA polymerases also can carry out a faithful and processive DNA replication. He also performing structural-function studies and analysis of the influence of metal cofactor during the process.\nFind him on Researchgate,  @CarlosDOC_ or by e-mail: cordonez@cbm.csic.es\n\n\nMario Rodríguez Mestre (PhD Student, 2020-2021 )\n\n\n​Liubov Chuprikova (Bioinformatics MSc, 2019-2020)\nLiuba developed ExplorePipolin, a pipeline for the annotation and characterization of pipolins. Her Master thesis was carried out under the joint supervision of Modesto and María de Toro, head of the Genomics and Bioinformatics facility of CIBIR, and an expert in the annotation of prokaryotic genomes and mobilome. We are grateful to Liuba’s help after her Thesis, which allowed us to publish ExplorePipolin in 2022.\n\n\nIrene Díaz García (BSc Student, 2020-2021)\n\n\nLorenzo Vargas Román (BSc Student, 2020-2021)\n\n\nAna Lechuga Mateo (MSc and PhD Student, 2016-2020)\nAna carried out her MSc in Virology in Salas’ lab under Modesto’s supervision and then she decided to stay in the lab for Ph.D. She worked in two different but complementary aspects of Bam35 tectivirus biology. First, she is characterizing viral DNA binding proteins and their role in TP-DNA genome replication. Moreover, she has been the main responsible for the VirHost-omics project, using a Y2H screen and next-generation sequence to study virus-host relationships by high-through"
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    "section": "The lab in September 2021",
    "text": "The lab in September 2021"
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    "title": "DNA polymerases",
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    "text": "DNA Polymerase (DNAP)\n\n\n\nDNAPs are our favorite enzymes. In our lab, we analyze their amino acid sequences in order to gain insights into their origin and evolution.\nWe also analyze their biochemical properties and biological role in vivo. We also try to infer possible applications in biotechnology and biomedicine.\nMany applications with fundamental importance in modern molecular biology and biomedicine, including the polymerase chain reaction (PCR) and whole genome DNA amplification (WGA) as well as some of the state-of-the-art DNA sequencing technologies, would not be feasible without the advances made in characterizing DNA polymerases (DNAPs) during the last 60 years. Furthermore, the development of WGA at the single-cell and single-molecule level has contributed to some of the most recent breakthroughs in our knowledge of different complex biological systems: from microbial ecosystems, shedding light into the microbial dark matter, to human disease, enhancing the sensitivity to detect genetic variants and mutation profiles of individual cells in a tissue or tumor and changing paradigms in early diagnosis of cancer and genetic diseases with non-invasive genetic tests.\nDNAPs are enzymes that synthesize DNA, by copying a pre-existing parental DNA molecule. Thus, they are responsible for preserving genetic information by replicating and repairing nucleic acid molecules in the cells. Their structure resembles a half-open hand, comprising the palm, thumb and fingers subdomains, which are arranged as a right hand in most of the DNA polymerase families (A, B, C, D, Y and RT), whereas members of the X family can be considered as left-handed. The single-subunit DNA-dependent RNA polymerases (DdRps) that are related to phage T7 RNA polymerase, and viral RNA- dependent polymerases (RdRps) also display a right-hand folding. A common mechanism and evolutionary origin for DNA and those RNA polymerases have been often suggested (Steitz et al. 1994, Koonin 2006, Monttinen et al. 2014); on the other hand, bifunctional primases-polymerases from archaeo-eukaryotic primases superfamily (AEPs) display an RNA-recognition motif fold with very distant similarities with DNA polymerases, suggesting a convergent evolution mechanism (Iyer et al. 2005, Guilliam et al. 2015)."
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    "text": "Although we always acknowledge our funding in publications and presentations, we also want to thank again our financiers and keep record of lab grants in this site. Unless otherwise stated, Modesto was the principal investigator of the grants below.\n\nFunctional characterization of primer-independent DNA polymerases in the context of genotoxic stress in bacteria\nFounder: Agencia Estatal de Investigación, 1/9/2022-31/8/2025\nGrant Ref. PID2021-123403NB-I00.\nFunding: 145.200€\n\n\n\nInsights into Pipolins diversity and dynamics in a wide range of pathogenic bacteria.\nFounder: UAM-CAM, 1/1/2022-31/12/2023\nGrant Ref. SI3-PJI-2021-00271.\nFunding: 42.120€.\n\n\n\nComprehensive virus-host protein interactome by the use of yeast-two-hybrid system coupled to next-generation sequencing analysis (VirHost-omics).\nFounder: Fundación Ramón Areces, 1/4/2019-31/12/2022 (PI since December 2019).\nGrant Ref.: CIVP19A5940.\nFunding: 129.600€\n\n\n\nPrimer-independent DNA polymerases and their applications in biotechnology and biomedicine.\nFounder: Agencia Estatal de Investigación, 1/1/2019-30/09/2022\nGrant Ref.: PGC2018-093723-A-I00.\nFunding: 127.050€\n\n\n\nNew fusion DNA polymerases with biotechnology applications.\nFounder: Fundación General CSIC, 1/9/2015-31/8/2018\nGrant Ref.: NewPols4Biotech.\nTotal Funding: 159.000€"
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    "text": "Lab pictures\n\n\n\nDecember 2023\n\n\n\n\n\n\n\nThe lab in September 2021"
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    "title": "DNA polymerases",
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    "text": "PolB\nAmong DNAPs, family B DNA polymerases (PolBs) have been suggested to be the most ancient group of DNA polymerases (Koonin 2006) and were usually divided into two groups (Filee et al. 2002): RNA-primed (rPolBs) and protein-primed (pPolBs). The rPolB group comprises mainly replicases devoted to accurate and efficient copying of large cellular and viral genomes, whereas pPolBs are exclusive to viruses, like Adenoviruses or bacteriophages from podovirus and tectivirus families, and self-replicating mobile elements with moderately-sized linear genomes (<50 kb) (Kazlauskas and Venclovas 2011, Krupovic and Koonin 2015).\n\nNew pPolBs and new DNA replication models\nBiochemical characterization of new PolBs and pPolBs beyond the classic models is very limited. However, the establishment of new models is required to explore the evolution as well as the potential of DNA polymerases. During the last years of Modesto in the Salas’ lab he and Margarita were lucky to recruit a couple of very good students that help to establish the Bacillus thuringiensis virus Bam35 as a new model for DNA replication. Like the virus Φ29, largely studied by Prof. Salas and her co-workers, Bam35 has a double stranded linear DNA genome, capped with a terminal protein on its 5’-ends.\nIn two consecutive papers, Berjón-Otero et al. 2015 and Berjón-Otero et al. 2016, the main characteristic of Bam35 DNA replication machinery were revealed. Briefly, Bam35 pPolB is a highly processive replicase endowed with translasion synthesis capacity opposite to abasic sites. Addtionally, full-length Bam35 TP-DNA can be replicated using only the viral TP and DNA polymerase and genome replication priming entails the TP deoxythymidylation at conserved tyrosine 194 and that this reaction is directed by the third base of the template strand. the genetic information of the first nucleotides of the genome can be recovered by a novel single-nucleotide jumping-back mechanism. Given the similarities between genome inverted terminal repeats and the genes encoding the replication proteins, we propose that related tectivirus genomes can be replicated by a similar mechanism, although replication of more distant genomes undergo by different process.\n\n\n\nGenome end sequences (A) and schematic representation of early replication steps (B) of representative viruses that replicate by a protein-priming mechanism. From Berjón-Otero et al. 2016\n\n\n\n\nEngineered enzymes\nThe characterization of B35DNAP prompted us to consider new applications of…\n\n\npiPolB\nIn collaboration with Patrick Forterre and Mart Krupovic (Pasteur Institute), we reported a third major group of PolBs, previously overlooked, named primer- independent PolBs (piPolBs), which display templated, de novo DNA synthesis capacity (Redrejo-Rodríguez et al. 2017). Contrary to RdRPs (Luo et al. 2000, van Dijk et al. 2004), DNAPs were believed to be unable to initiate replication de novo, which could be only partly justified with incomplete arguments, like the existence of hindrance impediments of dNTPs as compared with NTPs, the requirement for major protein structural modifications, or incompatibility with the proofreading activity (Lipps et al. 2003, Monttinen et al. 2014). Thus, the discovery of piPolBs dismisses those arguments and breaks the long-standing “primer rule”, a dogma in the field for 60 years, that stated that DNA polymerases required a pre-existing 3’-OH end to anchor the incoming nucleotide.\nThe evolutionary relationship among the three PolB groups is unknown and it is thus unclear whether the putative ancestral enzyme would have employed a primer and its nature (protein or RNA). Available phylogenetic analyses suggest that pPolBs and piPolBs might share a common ancestor (Figure 1C). Both groups, pPolBs, and piPolBs share the presence of specific subdomains, named TPR1 and TPR2 (Figure 1A), which were originally described in bacteriophage Φ29 pPolB (Φ29DNAP). TPR1 is required for the DNAP interaction with the TP and the DNA template strand, whereas TPR2 endows pPolB with the processivity and strand-displacement capacities (Salas et al. 2016). Indeed, the presence of TPR1 and TPR2 motifs have been usually a hallmark of pPolBs, which, provided that TPs sequences are usually not conserved, leads to prediction of a protein-primed DNA replication mechanism for a number of viruses (Peng et al. 2007, Fischer and Suttle 2011, among others) and self- replicative integrative genetic elements (Kapitonov & Jurka 2006), yet without experimental characterization of those DNAPs.\n\n\n\nFigure 1 (Redrejo-Rodríguez et al. 2017)\n\n\nThe piPolB-encoding genes are the hallmark of a new group of self-replicating mobile genetic elements (MGEs), that we named pipolins (for piPolB-encoding mobile element). Pipolins are integrated within the genomes of three highly diverse bacterial phyla (Firmicutes, Actinobacteria and Proteobacteria) and are also carried by mitochondria as circular plasmids. Multiple sequence analysis (MSA) showed that piPolB share exonuclease and polymerase motifs of PolBs, albeit with notable variations within the PolC and KxY motifs (Figure 1B)."
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    "section": "Bacterial RTs",
    "text": "Bacterial RTs\nReverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs.\nIn a recent work, in collaboration with the lab of Prof. Nicolás Toro, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential."
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    "text": "Contact us\nYou can reach us on Twitter @RnR_Lab\nFor inquires about publications or internships, please email Modesto (mredrejo at iib.uam.es)"
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    "section": "The lab By Metro",
    "text": "The lab By Metro\nThe lab is 10 min walk from the Begoña Metro Station (Exit to San Modesto). Follow the dashed line to the Biochemistry Department door. The arrow indicates a shortcut using a small door to the Medicine Campus.\nOnce inside the Campus walk straight until you find the big building of the School of Medicine."
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    "text": "Disclaimers\n\n\n\nThis section contains slides and other materials elaborated by lab members. Unless otherwise stated, all the materials are shared under CC BY-NC-SA license.\nAlthough most of the material is in English, some of the information of the courses may be in Spanish.\nTo optimize the site space, only the most recent versions are included using\ntemporal links. Please let us know if you cannot download some files."
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    "section": "Macromolecules Biosynthesis (3rd year, Fall Semester)",
    "text": "Macromolecules Biosynthesis (3rd year, Fall Semester)\nIn this course, we provide a detailed background on the flow of genetic information. Since the year 2019-2020, Modesto lecture the first part of the course focused on DNA Replication & Repair.\n\n2022-2023 Course Syllabus\n2022-2023 Replication and Repair Slides"
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    "section": "Coding Tools for Biochemistry and Molecular Biology (4rd year, Fall Semester)",
    "text": "Coding Tools for Biochemistry and Molecular Biology (4rd year, Fall Semester)\nThis is an introductory course to bioinformatics coding, by the use of Python and R. Since the year 2021-22 Modesto lecture most of the R section.\n\n2022-2023 Syllabus\n2022-2023 Reference Materials\n2022-2023 Slides"
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    "section": "Replication, Repair and Genome’s Instability (Master’s Degree in Biomolecules and Cell Dynamics)",
    "text": "Replication, Repair and Genome’s Instability (Master’s Degree in Biomolecules and Cell Dynamics)\nRepair of oxidative DNA damage (Slides 2022-2023)."
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    "section": "CIVIS Bioinformatics for non-bioinformaticians (link)",
    "text": "CIVIS Bioinformatics for non-bioinformaticians (link)\nSummer School in Tübingen (18-22 July 2022)\nHands on Introduction to Protein Modeling"
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    "section": "Biosynthesis of Macromolecules (3rd year, Fall Semester)",
    "text": "Biosynthesis of Macromolecules (3rd year, Fall Semester)\nIn this course, we provide a detailed background on the flow of genetic information. Since the year 2019-2020, Modesto lecture the first part of the course focused on DNA Replication & Repair.\n\n2022-2023 Course Syllabus\n2022-2023 Replication and Repair Slides"
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    "section": "Experimental Models in Biomedicine (4rd year, Fall Semester)",
    "text": "Experimental Models in Biomedicine (4rd year, Fall Semester)\nIn this course, we mentor the students in developing research projects using diverse biological models. Namely, we participate in the use of yeast two-hybrid boosted by high-throughput sequencing approaches to study protein interactomes.\n\n2022-2023 Syllabus\n2022-2023 Slides"
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    "text": "Bioinformatics for non-bioinformaticians (link)\nCIVIS Summer School in Tübingen (18-22 July 2022).\nHands on Introduction to Protein Modeling"
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    "title": "Mobile Genetic Elements",
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    "text": "Acknowledgment\n\n\n\nThis brief section belongs to the Introduction of the Master’s Thesis from one of our students, Víctor Mateo-Cáceres (UAM, June 2022).\n\n\n\n\n\n\n\n\n\nWatch out! This page is still being synthesized!\n\n\n\n\n\n\nOver the course of evolution, genetic material has found diverse ways of mobilization beyond vertical inheritance. A whole spectrum of vehicles, ranging from plasmids to phages, is used by all bacterial species for the horizontal transference of DNA between cells Partridge et al. (2018). These wandering DNA sequences, referred to as Mobile Genetic Elements (MGE), can encode virulence determinants, antimicrobial resistance (AMR) genes, phage defense systems, and other factors that grant their hosts new phenotypic traits (khosts?’2020) . MGE transference is especially relevant in prokaryotes, where specific DNA mobilization systems have been developed to enhance and optimize the acquisition of these elements (Frost et al. 2005). In fact, obtaining advantageous features through MGE transference is the main form of short-term adaptation in bacteria and can become crucial for cell survival, as in the case of AMR genes. The concerning increase of multidrug-resistant (MDR) bacteria is a clear example of this last point (Magiorakos et al. 2012). Therefore, it is essential to discover and characterize new mechanisms of genetic transference in order to disclose which features allow certain bacterial strains to succeed over the rest, especially the pathogenic ones.\nHorizontal genetic exchange mechanisms can be classified into three main groups according to the DNA introduction pathway: transformation, transduction, and conjugation (Figure 1). Transformation is the direct uptake of genetic material from the extracellular medium, which is frequently inefficient and can require activating the competence state Firth et al. (2018). Transduction refers to the transport of foreign DNA by viral particles (Humphrey et al. 2021). Conjugation, which has been extensively studied in plasmids, involves the transference of DNA from a donor cell to a recipient cell through the Type IV Secretion System (T4SS) (Smillie et al. 2010).\n\n\n\nFigure 1. Main mechanisms of genetic horizontal transfer. (A) Transformation: direct extracellular DNA uptake by the cell. (B) Transduction: DNA transference through the injection of a phage genetic content. (C) Conjugation: DNA transference through the Type IV Secretion System. T4SS: Type IV Secretion System, T4CP: T4SS coupling protein. Modified with Biorender from Frost et. al (2005)\n\n\nConjugative plasmids typically carry all genes necessary for T4SS configuration plus a T4SS coupling protein (T4CP), which is involved in the T4SS-DNA interaction, and a relaxase, which is a nickase necessary for generating the single-stranded DNA (ssDNA) that will be transferred. In fact, relaxases can be divided into several families according to their sequence and have been proposed as markers for plasmid classification (Smillie et al. 2010; Garcillan-Barcia, Francia, and Cruz 2009). Nevertheless, we currently know conjugative plasmids that lack some of the conjugation modules and conjugative plasmids with the capacity of integrating into the genome, also called Integrative and Conjugative Elements (ICEs) (Botelho and Schulenburg 2021), remarking the high flexibility of this type of MGEs."
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    "text": "Pipolins, our model MGE\nApart from phages, plasmids, ICEs, and other MGEs that have been in the spotlight for the last years, such as integrons or CRISPR-Cas Associated Transposons (CASTs), one group recently discovered stands out for its large genetic diversity and variability: pipolins. This new superfamily of MGEs comprises elements found in the three major bacterial phyla and even mitochondria Redrejo-Rodríguez et al. (2017). The only gene shared by all pipolins encodes for a replicative DNA polymerase from the family B (PolB), granting these elements the category of the so-called “self-replicating” elements along with the abovementioned Polintons and Casposons. However, PolBs from pipolins do not need a DNA/RNA 3’-end or a protein as a primer to initiate the complementary strand synthesis, making pipolins a unique class of elements inside the MGE universe. Furthermore, the biochemical characterization of this polymerase named piPolB (from primer-independent) has shown that it has capacity of proofreading, strand displacement, and replication over both undamaged and damaged templates. All of these features plus the de-novo DNA synthesis suggest a possible role for the piPolB in pipolin replication and/or cell DNA damage tolerance. However, we could generate a E. coli strain harboring pipolin but lacking the piPolB, which indicates that piPolB is not essential for pipolin maintenance. This result raised questions about the biological role of piPolB, but also about the “self-replicating” GME. Are they replicating at all?\nLater on, we could characterize the pipolins in pathogenic E. coli strains, showing that they are highly flexible and diverse, with the piPolB and the integration site being the only features in common to all elements Flament-Simon et al. (2020). We described this work also in this post in Nature Microbiology Community site.\n\nE. coli pipolins appear as integrated elements next to a specific tRNA gene, delimited by two direct repetitions (DRs) similar to phage atts, and encode for several distinct functions other than the piPolB. Among these different functions, we often find: tyrosine-recombinases (Y-rec), probably responsible for the excision and integration of the element; restriction-modification (RM) enzymes, known as defense systems against foreign DNA; and many other genes related to DNA mobilization and metabolism (integrases, Uracil-DNA glycosylases, excisionases). Surprisingly, no antimicrobial resistance gene was found in any pipolin except very few cases."
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    "text": "ExplorePipolin\nThe recent accessibility of high-throughput sequencing methods as part of surveillance programs of bacterial pathogens allows the genomic and metagenomic monitoring of the expansion of bacterial strain-specific markers, including virulence and AMR genes. However, these fast-evolving methods also generated a burden of data that must be throughout processed and analyzed Mitchell and Simner (2019). This is particularly problematic regarding the study of dynamics and plasticity of MGEs, as they can range in size from very simple and small elements, such as insertion elements (IS), coding for only the transposase necessary for their relocation, to large prophages, transposons and plasmids, which can be tens or hundreds of kilobase pairs in length and also appear associated among themselves Partridge et al. (2018) Durrant et al. (2020) Benler et al. (2021). Further, MGEs prediction and analysis are hindered by their great modularity and rapid evolution through gene acquisition and gene loss. Many pipelines designed for the analysis of MGEs are specialized and rely on the identification of hallmark genes, like plasmid replication proteins Carattoli and Hasman (2020), relaxases Alvarado, Garcillán-Barcia, and Cruz (2012) or specific transposase or recombinases for integrative elements Ross et al. (2021) Siguier et al. (2012) Moura et al. (2009) Cury, Touchon, and Rocha (2017) Cury et al. (2016). Some works have focused on the use of selected, high-quality reference genomes, but at the cost of diversity loss Jiang et al. (2019).\nIn order to facilitate the characterization of pipolins, we developed ExplorePipolin, a Python-based pipeline that screens for the presence of the element and performs its reconstruction and annotation. The pipeline can be used on virtually any genome from diverse organisms and of diverse quality, obtaining the highest-scored possible structure and reconstructed out of different contigs if necessary. Then, predicted pipolin boundaries and pipolin encoded genes are subsequently annotated using a custom database, returning the standard file formats suitable for comparative genomics of this mobile element."
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    "text": "Watch out! This page is still being synthesized!\n\n\n\nIn the meantime, we suggest our recent review on DNA polymerases for whole (meta)genome amplification: https://mdpi.com/1422-0067/24/11/9331"
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    "text": "Tectiviruses as a model for protein-primed DNA replication\n\n\n\n\n\n\n\nWatch out! This page is still being synthesized!"
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    "text": "The lab By Metro\nThe lab is 10 min walk from the Begoña Metro Station (Exit to San Modesto). Follow the dashed line to the Biochemistry Department door. The arrow indicates a shortcut using a small door to the Medicine Campus.\nOnce inside the Campus walk straight until you find the big building of the School of Medicine."
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    "text": "Contact us\nYou can reach us on Twitter @RnR_Lab\nFor inquires about publications or internships, please email Modesto (mredrejo at iib.uam.es)"
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    "text": "It took some time, but our new method for whole genome and metagenomes DNA amplification is just published in NAR Genomics and Bioinformatics: https://academic.oup.com/nargab/article/5/3/lqad073/7246555\n\n\n\n\n\nThis work was led by @CarlosDOC_, with the participation of @karmayoral from the lab. We were also very fortunate to be able to count on the collaboration of Dr. Conceiçao Egas from Biocant-CNC (Portugal).\nIn this work, we used the piPolB to develop new methods for whole genome and metagenome amplification (WGA). We used the piPolB in two different protocols: (1) piPolB MDA and (2) piMDA, with piPolB in combination with a Φ29 DNA polymerase (Φ29DNAP). In short, the second proved to be a great protocol, outperforming all previously available methods. In particular, our piMDA method (piPolB + Φ29DNAP) provides not only high DNA yield but also a very competent and unbiased coverage for sequences with high GC content, usually the Achilles heel of most MDA methods.\nAlong the way, we found that piPolB is capable of ab initio DNA synthesis, without DNA primers or templates. This activity has been described before for other polymerases, especially thermoresistant enzymes, but it is often neglected in the literature. Ab initio DNA synthesis by piPolB is negligible for optimized piMDA methods, but is of importance for piPolB solo amplifications. Ongoing work aims to understand how this spurious DNA synthesis is regulated and control it to developing improved piPolB-base MDA methodologies.\nWe have performed deep sequencing and a detailed comparison of non-amplified samples with samples that were amplified with piPolB MDA and piMDA, with and without a previous alkaline denaturation step. Additionally, we compared the piPolB-based methods with two commercially available kits based on Φ29DNAP, namely RepliG (Qiagen) for Random-Primers MDA and TruePrime (4BaseBio) for a primase-based MDA.\nAll in all, we can conclude that piMDA methods enable proficient WGA of a wide range of genomes for downstream applications, including those related to the study of microbiome diversity in different environments, especially in environments where high-GC microorganisms, such as halophiles or thermophiles, would predominate. In addition, our results suggest that piMDA has great potential for application in microbiome studies involving DNA amplification, such as those using single-cell metagenomics to reconstruct strain-resolved genomes of microbial communities at once, at the risk of missing poorly represented sequences with high GC content.\nFinally, we would like to dedicate this work to the memory of Professor Margarita Salas, for her long and inspiring support in our careers and for her seminal contributions to the discovery of piPolB and the early development of this project.\n\n\n\n\n\n\nThe WIPO site publishes today the decision to grant the European Patent of piPolB, entitled “Primer-independent DNA polymerases and their use for DNA synthesis”, with ref. EP305029628.\n\n\n\n\n\n\nWe are happy to share our new review on DNA polymerases for whole (meta)genome amplification: https://mdpi.com/1422-0067/24/11/9331 by @CarlosDOC_ and @mredrejo\n\n\n\n\n\n\nModesto was one of the lectures in this great summer school @uni_tue, hosted by Profs. Thorsten Schmidt and Andre F. Martins. \n\n\n\nBehind the paper in Nature Microbiology Community. Link"
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    "text": "We are happy to share our new review on DNA polymerases for whole (meta)genome amplification: https://mdpi.com/1422-0067/24/11/9331 by @CarlosDOC_ and @mredrejo\n\n\n\n\n\n\nModesto was one of the lectures in this great summer school @uni_tue, hosted by Profs. Thorsten Schmidt and Andre F. Martins.\n\n\n\n\nBehind the paper in Nature Microbiology Community. Link"
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    "text": "It took some time, but our new method for whole genome and metagenomes DNA amplification is just published in NAR Genomics and Bioinformatics: https://academic.oup.com/nargab/article/5/3/lqad073/7246555\n\n\n\n\n\nThis work was led by @CarlosDOC_, with the participation of @karmayoral from the lab. We were also very fortunate to be able to count on the collaboration of Dr. Conceiçao Egas from Biocant-CNC (Portugal).\nIn this work, we used the piPolB to develop new methods for whole genome and metagenome amplification (WGA). We used the piPolB in two different protocols: (1) piPolB MDA and (2) piMDA, with piPolB in combination with a Φ29 DNA polymerase (Φ29DNAP). In short, the second proved to be a great protocol, outperforming all previously available methods. In particular, our piMDA method (piPolB + Φ29DNAP) provides not only high DNA yield but also a very competent and unbiased coverage for sequences with high GC content, usually the Achilles heel of most MDA methods.\nAlong the way, we found that piPolB is capable of ab initio DNA synthesis, without DNA primers or templates. This activity has been described before for other polymerases, especially thermoresistant enzymes, but it is often neglected in the literature. Ab initio DNA synthesis by piPolB is negligible for optimized piMDA methods, but is of importance for piPolB solo amplifications. Ongoing work aims to understand how this spurious DNA synthesis is regulated and control it to developing improved piPolB-base MDA methodologies.\nWe have performed deep sequencing and a detailed comparison of non-amplified samples with samples that were amplified with piPolB MDA and piMDA, with and without a previous alkaline denaturation step. Additionally, we compared the piPolB-based methods with two commercially available kits based on Φ29DNAP, namely RepliG (Qiagen) for Random-Primers MDA and TruePrime (4BaseBio) for a primase-based MDA.\nAll in all, we can conclude that piMDA methods enable proficient WGA of a wide range of genomes for downstream applications, including those related to the study of microbiome diversity in different environments, especially in environments where high-GC microorganisms, such as halophiles or thermophiles, would predominate. In addition, our results suggest that piMDA has great potential for application in microbiome studies involving DNA amplification, such as those using single-cell metagenomics to reconstruct strain-resolved genomes of microbial communities at once, at the risk of missing poorly represented sequences with high GC content.\nFinally, we would like to dedicate this work to the memory of Professor Margarita Salas, for her long and inspiring support in our careers and for her seminal contributions to the discovery of piPolB and the early development of this project.\n\n\n\n\n\n\nThe WIPO site publishes today the decision to grant the European Patent of piPolB, entitled “Primer-independent DNA polymerases and their use for DNA synthesis”, with ref. EP305029628.\n\n\n\n\n\n\nWe are happy to share our new review on DNA polymerases for whole (meta)genome amplification: https://mdpi.com/1422-0067/24/11/9331 by @CarlosDOC_ and @mredrejo\n\n\n\n\n\n\nModesto was one of the lectures in this great summer school @uni_tue, hosted by Profs. Thorsten Schmidt and Andre F. Martins. \n\n\n\nBehind the paper in Nature Microbiology Community. Link"
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