Homologous Recombination In Bacteria Research Articles

Structural insights into the mechanism of DNA branch migration during homologous recombination in bacteria.

Some DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination.

Does selection favour the maintenance of porous species boundaries?

The endpoint of speciation has been viewed as complete isolation and the absence of gene flow between species. If the influx of genes from another species is maladaptive because species have different adaptations and genetic backgrounds, selection should favour the closing of species boundaries and zero gene flow, a process known as reinforcement. Recently, numerous cases of gene flow between species have been identified, many of which involved adaptive introgression of beneficial alleles. These cases could reflect transient states on the way to closed species boundaries or the result of declining strength or efficacy of selection for reinforcement as the level of gene flow approaches zero. An alternative hypothesis, however, is that selection favours porous species boundaries that allow beneficial alleles to cross, especially in changing environments. This perspective evaluates the conditions that would be needed for selection to favour porous species boundaries and the evidence for them. A contrast is made between hybridization in sexual eukaryotes and gene transfer via homologous recombination in bacteria. Current evidence is inconclusive on whether non-zero gene flow is favoured by selection. Studies are needed that quantify selection gradients on rates of gene flow and test for evolution towards intermediate values, especially experiments that manipulate conditions and track evolution for multiple generations. Estimation of gene flow networks for more clades and regional assemblages using emerging genome data will also allow the evolutionary determinants of interspecific gene flow to be better understood.

Structural and Biochemical Analysis of the Recombination Mediator Protein RecR from Campylobacter jejuni.

The recombination mediator complex RecFOR, consisting of the RecF, RecO, and RecR proteins, is needed to initiate homologous recombination in bacteria by positioning the recombinase protein RecA on damaged DNA. Bacteria from the phylum Campylobacterota, such as the pathogen Campylobacter jejuni, lack the recF gene and trigger homologous recombination using only RecR and RecO. To elucidate the functional properties of C. jejuni RecR (cjRecR) in recombination initiation that differ from or are similar to those in RecF-expressing bacteria, we determined the crystal structure of cjRecR and performed structure-based binding analyses. cjRecR forms a rectangular ring-like tetrameric structure and coordinates a zinc ion using four cysteine residues, as observed for RecR proteins from RecF-expressing bacteria. However, the loop of RecR that has been shown to recognize RecO and RecF in RecF-expressing bacteria is substantially shorter in cjRecR as a canonical feature of Campylobacterota RecR proteins, indicating that cjRecR lost a part of the loop in evolution due to the lack of RecF and has a low RecO-binding affinity. Furthermore, cjRecR features a larger positive patch and exhibits substantially higher ssDNA-binding affinity than RecR from RecF-expressing bacteria. Our study provides a framework for a deeper understanding of the RecOR-mediated recombination pathway.

Speeding up Inference of Homologous Recombination in Bacteria

Bacteria reproduce clonally but most species recombine frequently, so that the ancestral process is best captured using an ancestral recombination graph. This graph model is often too complex to be used in an inferential setup, but it can be approximated for example by the ClonalOrigin model. Inference in the ClonalOrigin model is performed via a Reversible-Jump Markov Chain Monte Carlo algorithm, which attempts to jointly explore: the recombination rate, the number of recombination events, the departure and arrival points on the clonal genealogy for each recombination event, and the range of genomic sites affected by each recombination event. However, the Reversible-Jump algorithm often performs poorly due to the complexity of the target distribution since it needs to explore spaces of different dimensions. Recent developments in Bayesian computation methodology have provided ways to improve existing methods and code, but are not well-known outside the statistics community. We show how exploiting one of these new computational methods can lead to faster inference under the ClonalOrigin model.

Single-molecule characterization of compressed RecA nucleoprotein filaments

RecA is a central enzyme of homologous recombination in bacteria, which plays a major role in DNA repair, natural transformation and SOS-response activation. RecA forms nucleoprotein filaments on single-stranded DNA with a highly conserved architecture that is also shared by eukaryotic recombinases. One of the key features of these filaments is the ability to switch between stretched and compressed conformations in response to ATP binding and hydrolysis. However, the functional role of such conformational changes is not fully understood. Structural data revealed that in the absence of ATP RecA binds DNA with the stoichiometry of 5 nucleotides per one monomer, while in the presence of ATP the binding stoichiometry is 3:1. Such differences suggest incompatibility of the active and inactive conformations, yet dynamic single-molecule studies demonstrated that ATP and apo conformations can be directly interconvertible. In the present work we use a single-molecule approach to address the features of inactive RecA nucleoprotein filaments formed de novo in the absence of nucleotide cofactors. We show that compressed RecA-DNA filaments can exist with both 5:1 and 3:1 binding stoichiometry which is determined by conditions of the filament assembly. However, only a 3:1 stoichiometry allows direct interconvertibility with the active ATP-bound conformation.

Expression pattern of the V5-Ostm1 protein in bacterial artificial chromosome transgenic mice.

Mutations in the osteopetrotic transmembrane protein 1 (Ostm1) gene are responsible for the most severe form of autosomal recessive osteopetrosis both in humans and in the gray lethal (gl/gl) mouse. This defect leads to increased bone mass with bone marrow occlusion and hematopoietic defects. To establish the expression profile of the mouse Ostm1 protein in vivo, homologous recombination in bacteria was designed to generate a V5-Ostm1 bacterial artificial chromosome (BAC) that was subsequently integrated in the mouse genome. Tissue expression of the transgene V5-Ostm1 RNA and protein in transgenic mice follow the endogenous expression profile. Immunohistochemistry analysis demonstrated expression in neuronal populations from central and peripheral nervous system and defined a unique cellular expression pattern. Importantly, together with appropriate protein post-translational modification, in vivo rescue of the osteopetrotic bone gl/gl phenotype in BAC V5-Ostm1 gl/gl mice is consistent with the expression of a fully functional and active protein. These mice represent a unique tool to unravel novel Ostm1 functions in individual tissue and neuronal cell populations and the V5-Ostm1 transgene represents an easy visual marker to monitor the expression of Ostm1 in vitro and in vivo.

Inferring bacterial recombination rates from large-scale sequencing datasets.

We present a robust, computationally efficient method ( https://github.com/kussell-lab/mcorr ) for inferring the parameters of homologous recombination in bacteria, which can be applied in diverse datasets, from whole-genome sequencing to metagenomic shotgun sequencing data. Using correlation profiles of synonymous substitutions, we determine recombination rates and diversity levels of the shared gene pool that has contributed to a given sample. We validated the recombination parameters using data from laboratory experiments. We determined the recombination parameters for a wide range of bacterial species, and inferred the distribution of shared gene pools for global Helicobacter pylori isolates. Using metagenomics data of the infant gut microbiome, we measured the recombination parameters of multidrug-resistant Escherichia coli ST131. Lastly, we analyzed ancient samples of bacterial DNA from the Copper Age 'Iceman' mummy and from 14th century victims of the Black Death, obtaining measurements of bacterial recombination rates and gene pool diversity of earlier eras.

Transgene Recombineering in Bacterial Artificial Chromosomes.

Bacterial Artificial Chromosome (BAC) libraries are a valuable research resource. Any oneof the clones in these libraries can carry hundreds of thousands of base pairs of genetic information. Often the entire coding sequence and significant upstream and downstream regions, including regulatory elements, can be found in a single BAC clone. BACs can be put to many uses, such as to study the function of human genes in knockout mice, to drive reporter gene expression in transgenic animals, and for gene discovery. In order to use BACs for experimental purposes it is often desirable to genetically modify them by introducing reporter elements or heterologous cDNA sequences. It is not feasible to use conventional DNA cloning approaches to modify BACs due to their size and complexity, thus a specialized field "recombineering" has developed to modify BAC clones through the use of homologous recombination in bacteria with short homology regions. Genetically engineered BACs can then be used in cell culture, mouse, or rat models to study cancer, neurology, and genetics.

Oncolytic adenovirus Ad657 for systemic virotherapy against prostate cancer.

BackgroundHuman species C adenovirus serotype 5 (Ad5) is the archetype oncolytic adenovirus and has been used in the vast majority of preclinical and clinical tests. While Ad5 can be robust, species C Ad6 has lower seroprevalence, side effects, and appears to be more potent as a systemic therapy against a number of tumors than Ad5. Historically, there have only been four species C human adenoviruses: serotypes 1, 2, 5, and 6. More recently a new species C adenovirus, Ad57, was identified. Ad57 is most similar to Ad6 with virtually all variation in their capsid proteins occurring in the hypervariable regions (HVRs) of their hexon proteins. Most adenovirus neutralizing antibodies target the HVRs on adenoviruses. This led us to replace the hexon HVRs in Ad6 with those from Ad57 to create a new virus called Ad657 and explore this novel species C platform’s utility as an oncolytic virus.MethodsThe HVR region from Ad57 was synthesized and used to replace the Ad6 HVR region by homologous recombination in bacteria generating a new viral platform that we call Ad657. Replication-competent Ad5, Ad6, and Ad657 were compared in vitro and in vivo for liver damage and oncolytic efficacy against prostate cancers after single intravenous treatment in mice.ResultsAd5, Ad6, and Ad657 had similar in vitro oncolytic activity against human prostate cancer cells. Ad5 provoked the highest level of liver toxicity after intravenous injection and Ad657 caused the least damage in mice. Previous data demonstrated that Ad6 was superior to Ad5 at killing distant subcutaneous prostate cancer tumors in mouse models after a intravenous injection. Given this, Ad657 was compared to the Ad6 benchmark virus by single intravenous injection into mice bearing subcutaneous human DU145 prostate cancers. Under these conditions, Ad657 first infected the liver and then reached distant tumors. Both Ad6 and Ad657 mediated significant delays in tumor growth and extension of survival with Ad6 mediating higher efficacy.ConclusionsThese data suggest that Ad657 may have utility as a local or systemic oncolytic virotherapy for prostate cancers. These data also lay the foundation for serotype-switching with oncolytic species C Ads.

A One-Step System for Convenient and Flexible Assembly of Transcription Activator-Like Effector Nucleases (TALENs).

Transcription activator-like effector nucleases (TALENs) are powerful tools for targeted genome editing in diverse cell types and organisms. However, the highly identical TALE repeat sequences make it challenging to assemble TALEs using conventional cloning approaches, and multiple repeats in one plasmid are easily catalyzed for homologous recombination in bacteria. Although the methods for TALE assembly are constantly improving, these methods are not convenient because of laborious assembly steps or large module libraries, limiting their broad utility. To overcome the barrier of multiple assembly steps, we report a one-step system for the convenient and flexible assembly of a 180 TALE module library. This study is the first demonstration to ligate 9 mono-/dimer modules and one circular TALEN backbone vector in a one step process, generating 9.5 to 18.5 repeat sequences with an overall assembly rate higher than 50%. This system makes TALEN assembly much simpler than the conventional cloning of two DNA fragments because this strategy combines digestion and ligation into one step using circular vectors and different modules to avoid gel extraction. Therefore, this system provides a convenient tool for the application of TALEN-mediated genome editing in scientific studies and clinical trials.

RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens.

Chloroplast DNA (cpDNA) encodes essential genes for chloroplast functions, including photosynthesis. Homologous recombination occurs frequently in cpDNA; however, its significance and underlying mechanism remain poorly understood. In this study, we analyzed the role of a nuclear-encoded chloroplast-localizedhomolog of RecA recombinase, which is a key factor in homologous recombination in bacteria, in the moss Physcomitrella patens. Complete knockout (KO) of the P.patens chloroplast RecA homolog RECA2 caused a modest growth defect and conferred sensitivity to methyl methanesulfonate and UV. The KO mutant exhibited low recovery of cpDNA from methyl methanesulfonate damage, suggesting that RECA2 knockout impairs repair of damaged cpDNA. The RECA2 KO mutant also exhibited reduced cpDNA copy number and an elevated level of cpDNA molecule resulting from aberrant recombination between short dispersed repeats (13-63bp), indicating that the RECA2 KO chloroplast genome was destabilized. Taken together, these data suggest a dual role for RECA2 in the maintenance of chloroplast genome stability: RECA2 suppresses aberrant recombination between short dispersed repeats and promotes repair of damaged DNA.

Deinococcus radiodurans RecA nucleoprotein filaments characterized at the single-molecule level with optical tweezers

Deinococcus radiodurans can survive extreme doses of ionizing radiation due to the very efficient DNA repair mechanisms that are able to cope even with hundreds of double-strand breaks. RecA, the critical protein of homologous recombination in bacteria, is one of the key components of the DNA-repair system. Repair of double-strand breaks requires RecA binding to DNA and assembly of the RecA nucleoprotein helical filaments. The Escherichia coli RecA protein (EcRecA) and its interactions with DNA have been extensively studied using various approaches including single-molecule techniques, while the D. radiodurans RecA (DrRecA) remains much less characterized. However, DrRecA shows some remarkable differences from E. coli homolog. Here we combine microfluidics and single-molecule DNA manipulation with optical tweezers to follow the binding of DrRecA to long double-stranded DNA molecules and probe the mechanical properties of DrRecA nucleoprotein filaments at physiological pH. Our data provide a direct comparison of DrRecA and EcRecA binding to double-stranded DNA under identical conditions. We report a significantly faster filaments assembly as well as lower values of persistence length and contour length for DrRecA nucleoprotein filaments compared to EcRecA. Our results support the existing model of DrRecA forming more frequent and less continuous filaments relative to those of EcRecA.

English

High-level expression of recombinant human blood coagulation factor in milk of farm animals at a large scale provides a powerful tool for protein production. However, a bottleneck in recent protein synthesis technologies is the high cost of current transgenic livestock system. Here, we report a simple, rapid, and low-cost protein production method based on a replication-defective adenoviral vector system. The recombinant hfVIII adenoviral vector was generated by using homologous recombination in bacteria and transfected the plasmids into the HEK293 packaging cell line. Goat’s mammary glands at the different physiological stage were infected with the recombinant adenovirus containing a human blood coagulation factor VIII gene. The expression level of bioactive hfVIII from milk sample was confirmed by Western blot analysis and ELISA methods. The hfVIII gene was expressed as a protein of about 26 kDa and no recombinant hfVIII protein was detected in negative control treatments. The hfVIII was expressed with a high activity level up to 1.4 U/ml in the milk of goats. These data suggest that the approach established here could be a low cost and further increase expression efficacy for the recombinant human blood coagulation factor and other biopharmaceutical interest. Key words: Adenovirus vector, human blood coagulation factor VIII, mammary gland, milk, goat, recombinant protein.

Homologous recombination using bacterial artificial chromosomes.

This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase ("ChAT"), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment.

Infectious delivery of alphaherpesvirus bacterial artificial chromosomes.

Bacterial artificial chromosomes (BACs) can accommodate and stably propagate the genomes of large DNA viruses in E. coli. As DNA virus genomes are often per se infectious upon transfection into mammalian cells, their cloning in BACs and easy modification by homologous recombination in bacteria has become an important strategy to investigate the functions of individual virus genes. This chapter describes a strategy to clone the genomes of viruses of the Alphaherpesvirinae subfamily within the family of the Herpesviridae, which is a group of large DNA viruses that can establish both lytic and latent infections in most animal species including humans. The cloning strategy includes the following steps: (1) Construction of a transfer plasmid that contains the BAC backbone with selection and screening markers, and targeting sequences which support homologous recombination between the transfer plasmid and the alphaherpesvirus genome. (2) Introduction of the transfer plasmid sequences into the alphaherpesvirus genome via homologous recombination in mammalian cells. (3) Isolation of recombinant virus genomes containing the BAC backbone sequences from infected mammalian cells and electroporation into E. coli. (4) Preparation of infectious BAC DNA from bacterial cultures and transfection into mammalian cells. (5) Isolation and characterization of progeny virus.

Pancreatic transduction by helper-dependent adenoviral vectors via intraductal delivery.

Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue.

Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells

Helicobacter pylori is a gram-negative pathogenic bacterium that colonises the human stomach. The chronic infection it causes results in peptic ulcers and gastric cancers. H. pylori can easily establish a chronic infection even if the immune system attacks this pathogen with oxidative stress agents and immunoglobulins. This is attributed to bacterial defence mechanisms against these stresses. As a defence mechanism against oxidative stresses, in bacterial genomes, homologous recombination can act as a repair pathway of DNA's double-strand breaks (DSBs). Moreover, homologous recombination is also involved in the antigenic variation in H. pylori. Gene conversion alters genomic structures of babA and babB (encoding outer membrane proteins), resulting in escape from immunoglobulin attacks. Thus, homologous recombination in bacteria plays an important role in the maintenance of a chronic infection. In addition, H. pylori infection causes DSBs in human cells. Homologous recombination is also involved in the repair of DSBs in human cells. In this review, we describe the roles of homologous recombination with an emphasis on the maintenance of a chronic infection.

Construction and identification of a recombinant adenovirus expressing bioactive GM-CSF: Implication for GM-CSF secreting tumor vaccines design

IntroductionGranulocyte-monocyte colony stimulating factor (GM-CSF) has been reported to to mediate antitumor effects through stimulation of various antigen presentation cells. Clearly, these properties make GM-CSF a potent adjuvant. Therefore, an oncolytic adenovirus coding for GMCSF was engineered. ObjectiveContraction, identification and characterization of recombinant adenovirus encoding GM-CSF. MethodsIn this study, a recombinant adenovirus expressing GM-CSF (rAd-GM) was constructed and characterized. Homologous recombination in bacteria followed by transfection of recombinant plasmid into a mammalian packaging cell line, viral production was conveniently followed by the gene incorporated into the viral backbone. Results & DiscussionWe also showed that the construct produced a 12 kDa recombinant protein which stimulated colony formation in bone marrow cells in vitro, indicating that the recombinant GM-CSF was biologically active. ConclusionFuture work will demonstrate the rAd-GM mediated antitumor immunity in rats with experimentally induced breast cancer.

Generation of islet β cell-specific Cre recombinase targeting vector by homologous recombination in bacteria

Objective To construct a knock-in targeting vector for expressing of Cre recombinase specifically in isletβ cell and provide the key material for knock-in mice with Crerecombinase expressed in isletβcells,providing a knock-out animal model for studying the function of isletβcells.Methods In our study,we constructed a knock-in targeting vector using the third exon of insulin 2(Ins2)as a target site withλphage Red recombination system.Through a first homologous recombination,we cloned an about 12kb genomic DNA fragment from the bacterial artificial chromosome(BAC)which contained Ins2genomic DNA into a low copy vector pBR322-2sthrough gap repair.Meantime,a mini-targeting vector containing internal ribosome entry site(IRES),DNA sequences encoding Cre recombinase and positive-negative-selection(PNS)gene was generated.After second recombination,the final Ins2-Cretargeting vector was generated.Results With the third exon of Ins2used as the target,we successfully constructed the knock-in targeting vector expressing Cre recombinase which was controlled by endogenous Ins2gene.Conclusion We have successfully constructed the knock-in targeting vector expressing Cre recombinase,it will provide an important material for creating animal model of Cre recombinase which is specially expressed in isletβcells.

Homologous Recombination-Enzymes and Pathways.

Homologous recombination is an ubiquitous process that shapes genomes and repairs DNA damage. The reaction is classically divided into three phases: presynaptic, synaptic, and postsynaptic. In Escherichia coli, the presynaptic phase involves either RecBCD or RecFOR proteins, which act on DNA double-stranded ends and DNA single-stranded gaps, respectively; the central synaptic steps are catalyzed by the ubiquitous DNA-binding protein RecA; and the postsynaptic phase involves either RuvABC or RecG proteins, which catalyze branch-migration and, in the case of RuvABC, the cleavage of Holliday junctions. Here, we review the biochemical properties of these molecular machines and analyze how, in light of these properties, the phenotypes of null mutants allow us to define their biological function(s). The consequences of point mutations on the biochemical properties of recombination enzymes and on cell phenotypes help refine the molecular mechanisms of action and the biological roles of recombination proteins. Given the high level of conservation of key proteins like RecA and the conservation of the principles of action of all recombination proteins, the deep knowledge acquired during decades of studies of homologous recombination in bacteria is the foundation of our present understanding of the processes that govern genome stability and evolution in all living organisms.