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International Journal of Molecular... Aug 2021species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of... (Review)
Review
species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T-DNA often contains, at its junctions with plant DNA, deletions of T-DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T-DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T-DNA integration. However, the involvement of specific plant DNA repair proteins and proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T-DNA integration into the plant genome.
Topics: Agrobacterium; Chromosomes, Plant; DNA Repair; DNA, Bacterial; DNA, Plant; Plants; Transformation, Genetic
PubMed: 34445162
DOI: 10.3390/ijms22168458 -
Microbiology Spectrum Oct 2014Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a... (Review)
Review
Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy.
Topics: Bacteriophage mu; DNA Transposable Elements; DNA, Viral; Recombination, Genetic; Viral Proteins; Virus Integration
PubMed: 26104374
DOI: 10.1128/microbiolspec.MDNA3-0007-2014 -
Bioscience Reports Mar 2023Prokaryotes use the adaptive immunity mediated via the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated (CRISPR-Cas) system for protection... (Review)
Review
Prokaryotes use the adaptive immunity mediated via the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated (CRISPR-Cas) system for protection against invading elements such as phages and plasmids. The immunity is achieved by capturing small DNA fragments or spacers from foreign nucleic acids (protospacers) and integrating them into the host CRISPR locus. This step of CRISPR-Cas immunity called 'naïve CRISPR adaptation' requires the conserved Cas1-Cas2 complex and is often supported by variable host proteins that assist in spacer processing and integration. Bacteria that have acquired new spacers become immune to the same invading elements when reinfected. CRISPR-Cas immunity can also be updated by integrating new spacers from the same invading elements, a process called 'primed adaptation'. Only properly selected and integrated spacers are functional in the next steps of CRISPR immunity when their processed transcripts are used for RNA-guided target recognition and interference (target degradation). Capturing, trimming, and integrating new spacers in the correct orientation are universal steps of adaptation to all CRISPR-Cas systems, but some details are CRISPR-Cas type-specific and species-specific. In this review, we provide an overview of the mechanisms of CRISPR-Cas class 1 type I-E adaptation in Escherichia coli as a general model for adaptation processes (DNA capture and integration) that have been studied in detail. We focus on the role of host non-Cas proteins involved in adaptation, particularly on the role of homologous recombination.
Topics: Escherichia coli; CRISPR-Cas Systems; Plasmids; Escherichia coli Proteins; DNA
PubMed: 36809461
DOI: 10.1042/BSR20221198 -
Trends in Genetics : TIG Jul 2023Insects are crucial for ecosystem health but climate change and pesticide use are driving massive insect decline. To mitigate this loss, we need new and effective... (Review)
Review
Insects are crucial for ecosystem health but climate change and pesticide use are driving massive insect decline. To mitigate this loss, we need new and effective monitoring techniques. Over the past decade there has been a shift to DNA-based techniques. We describe key emerging techniques for sample collection. We suggest that the selection of tools should be broadened, and that DNA-based insect monitoring data need to be integrated more rapidly into policymaking. We argue that there are four key areas for advancement, including the generation of more complete DNA barcode databases to interpret molecular data, standardisation of molecular methods, scaling up of monitoring efforts, and integrating molecular tools with other technologies that allow continuous, passive monitoring based on images and/or laser imaging, detection, and ranging (LIDAR).
Topics: Animals; Ecosystem; Biodiversity; DNA Barcoding, Taxonomic; DNA; Insecta
PubMed: 36907721
DOI: 10.1016/j.tig.2023.02.012 -
Molecular Microbiology May 2024The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that... (Review)
Review
The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.
Topics: Bacteriophage lambda; Recombination, Genetic; DNA, Viral; Viral Proteins; DNA, Bacterial; Binding Sites; Integration Host Factors
PubMed: 38372210
DOI: 10.1111/mmi.15241 -
Microbiology Spectrum Apr 2015DNA transposases use a limited repertoire of structurally and mechanistically distinct nuclease domains to catalyze the DNA strand breaking and rejoining reactions that... (Review)
Review
DNA transposases use a limited repertoire of structurally and mechanistically distinct nuclease domains to catalyze the DNA strand breaking and rejoining reactions that comprise DNA transposition. Here, we review the mechanisms of the four known types of transposition reactions catalyzed by (1) RNase H-like transposases (also known as DD(E/D) enzymes); (2) HUH single-stranded DNA transposases; (3) serine transposases; and (4) tyrosine transposases. The large body of accumulated biochemical and structural data, particularly for the RNase H-like transposases, has revealed not only the distinguishing features of each transposon family, but also some emerging themes that appear conserved across all families. The more-recently characterized single-stranded DNA transposases provide insight into how an ancient HUH domain fold has been adapted for transposition to accomplish excision and then site-specific integration. The serine and tyrosine transposases are structurally and mechanistically related to their cousins, the serine and tyrosine site-specific recombinases, but have to date been less intensively studied. These types of enzymes are particularly intriguing as in the context of site-specific recombination they require strict homology between recombining sites, yet for transposition can catalyze the joining of transposon ends to form an excised circle and then integration into a genomic site with much relaxed sequence specificity.
Topics: DNA; DNA Transposable Elements; Recombination, Genetic; Transposases
PubMed: 26104718
DOI: 10.1128/microbiolspec.MDNA3-0034-2014 -
Microbiology Spectrum Apr 2015Although most of non-long terminal repeat (non-LTR) retrotransposons are incorporated in the host genome almost randomly, some non-LTR retrotransposons are incorporated... (Review)
Review
Although most of non-long terminal repeat (non-LTR) retrotransposons are incorporated in the host genome almost randomly, some non-LTR retrotransposons are incorporated into specific sequences within a target site. On the basis of structural and phylogenetic features, non-LTR retrotransposons are classified into two large groups, restriction enzyme-like endonuclease (RLE)-encoding elements and apurinic/apyrimidinic endonuclease (APE)-encoding elements. All clades of RLE-encoding non-LTR retrotransposons include site-specific elements. However, only two of more than 20 APE-encoding clades, Tx1 and R1, contain site-specific non-LTR elements. Site-specific non-LTR retrotransposons usually target within multi-copy RNA genes, such as rRNA gene (rDNA) clusters, or repetitive genomic sequences, such as telomeric repeats; this behavior may be a symbiotic strategy to reduce the damage to the host genome. Site- and sequence-specificity are variable even among closely related non-LTR elements and appeared to have changed during evolution. In the APE-encoding elements, the primary determinant of the sequence- specific integration is APE itself, which nicks one strand of the target DNA during the initiation of target primed reverse transcription (TPRT). However, other factors, such as interaction between mRNA and the target DNA, and access to the target region in the nuclei also affect the sequence-specificity. In contrast, in the RLE-encoding elements, DNA-binding motifs appear to affect their sequence-specificity, rather than the RLE domain itself. Highly specific integration properties of these site-specific non-LTR elements make them ideal alternative tools for sequence-specific gene delivery, particularly for therapeutic purposes in human diseases.
Topics: Binding Sites; DNA; Humans; Molecular Biology; Recombination, Genetic; Reverse Transcription; Short Interspersed Nucleotide Elements
PubMed: 26104700
DOI: 10.1128/microbiolspec.MDNA3-0001-2014 -
European Biophysics Journal : EBJ Sep 2022Nucleic acids' physical properties have been investigated by theoretical methods based both on fully atomistic representations and on coarse-grained models, e.g., the... (Review)
Review
Nucleic acids' physical properties have been investigated by theoretical methods based both on fully atomistic representations and on coarse-grained models, e.g., the worm-like-chain, taken from polymer physics. In this review article, I discuss an intermediate (mesoscopic) approach and show how to build a three-dimensional Hamiltonian model which accounts for the main interactions responsible for the stability of the helical molecules. While the 3D mesoscopic model yields a sufficiently detailed description of the helix at the level of the base pair, it also allows one to predict the thermodynamical and structural properties of molecules in solution. Relying on the idea that the base pair fluctuations can be conceived as trajectories, I have built over the past years a computational method based on the time-dependent path integral formalism to derive the partition function. While the main features of the method are presented, I focus here in particular on a newly developed statistical method to set the maximum amplitude of the base pair fluctuations, a key parameter of the theory. Some applications to the calculation of DNA flexibility properties are discussed together with the available experimental data.
Topics: Base Pairing; DNA; Molecular Structure; Nucleic Acid Conformation
PubMed: 35976412
DOI: 10.1007/s00249-022-01614-z -
Communications Biology Jul 2023Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular...
Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development.
Topics: Humans; Hepatitis B virus; Carcinoma, Hepatocellular; Liver Neoplasms; DNA, Mitochondrial; Virus Integration; Mitochondria
PubMed: 37400627
DOI: 10.1038/s42003-023-05017-4 -
European Biophysics Journal : EBJ Nov 2023Nucleic acids are highly deformable helical molecules constantly stretched, twisted and bent in their biological functioning. Single molecule experiments have shown that...
Nucleic acids are highly deformable helical molecules constantly stretched, twisted and bent in their biological functioning. Single molecule experiments have shown that double stranded (ds)-RNA and standard ds-DNA have opposite twist-stretch patterns and stretching properties when overwound under a constant applied load. The key structural features of the A-form RNA and B-form DNA helices are here incorporated in a three-dimensional mesoscopic Hamiltonian model which accounts for the radial, bending and twisting fluctuations of the base pairs. Using path integral techniques which sum over the ensemble of the base pair fluctuations, I compute the average helical repeat of the molecules as a function of the load. The obtained twist-stretch relations and stretching properties, for short A- and B-helical fragments, are consistent with the opposite behaviors observed in kilo-base long molecules.
Topics: Nucleic Acids; Nucleic Acid Conformation; DNA; Base Pairing; RNA
PubMed: 37357224
DOI: 10.1007/s00249-023-01669-6