Recombinase Family Research Articles

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

Comparative analysis of structural dynamics and allosteric mechanisms of RecA/Rad51 family proteins: Integrated atomistic MD simulation and network-based analysis

Homologous recombination plays a key role in double-strand break repair, stalled replication fork repair, and meiosis. The RecA/Rad51 family recombinases catalyze the DNA strand invasion reaction that occurs during homologous recombination. However, the high sequence differences between homologous groups have hindered the thoroughly studies of this ancient protein family. The dynamic mechanisms of the family, particularly at the residual level, remain poorly understood. In this work, five representative RecA/Rad51 recombinase family members from all major kingdoms of living organisms: prokaryotes, eukaryotes, archaea, and viruses, were selected to explore the molecular mechanisms behind their conserved biological significance. A variety of techniques, including all-atom molecular dynamics simulation, perturbation response scanning, and protein structure network analysis, were used to examine the flexibility and correlation of protein domains, distribution of sensors and effectors and conserved hub residues. Furthermore, the potential communication routes between the ATP-binding region and the DNA-binding region of each recombinase were identified. Our results demonstrate the conserved molecular dynamics of these recombinases in the early stage of homologous recombination, including cooperative motions between regions, conserved sensing and effecting functional residue distribution, and conserved hub residues. Meanwhile, the unique ATP-DNA communication routes of each recombinase was also revealed. These results provide new insights into the mechanism of RecA/Rad51 family proteins, and provide new theoretical guidance for the development of allosteric inhibitors and the application of RecA/Rad51 family proteins.

Functional characterization of Microplitis demolitor bracovirus genes that encode nucleocapsid components.

Understanding how bracoviruses (BVs) function in wasps is of broad interest in the study of virus evolution. This study characterizes most of the Microplitis demolitor bracovirus (MdBV) genes whose products are nucleocapsid components. Results indicate several genes unknown outside of nudiviruses and BVs are essential for normal capsid assembly. Results also indicate most MdBV tyrosine recombinase family members and the DNA binding protein p6.9-1 are required for DNA processing and packaging into nucleocapsids.

Computational Insights into the Dynamic Structural Features and Binding Characteristics of Recombinase UvsX Compared with RecA.

RecA family recombinases are the core enzymes in the process of homologous recombination, and their normal operation ensures the stability of the genome and the healthy development of organisms. The UvsX protein from bacteriophage T4 is a member of the RecA family recombinases and plays a central role in T4 phage DNA repair and replication, which provides an important model for the biochemistry and genetics of DNA metabolism. UvsX shares a high degree of structural similarity and function with RecA, which is the most deeply studied member of the RecA family. However, the detailed molecular mechanism of UvsX has not been resolved. In this study, a comprehensive all-atom molecular dynamics simulation of the UvsX protein dimer complex was carried out in order to investigate the conformational and binding properties of UvsX in combination with ATP and DNA, and the simulation of RecA was synchronized with the property comparison learning for UvsX. This study confirmed the highly conserved molecular structure characteristics and catalytic centers of RecA and UvsX, and also discovered differences in regional conformation, volatility and the ability to bind DNA between the two proteins at different temperatures, which would be helpful for the subsequent understanding and application of related recombinases.

Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases

Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.

Whole genome sequencing and annotation of a lysogenic phage vB_EcoP_DE5 isolated from donkey-derived Escherichia coli.

A lysogenic phage vB_EcoP_DE5 (hereafter designated DE5) was isolated from donkey-derived Escherichia coli. The bacteriophage was examined by transmission electron microscopy, and the result showed that DE5 belonged to the genus Kuravirus. DE5 was sensitive to changes in temperature and pH, and it could maintain its activity at pH 7 and below 60℃. The whole genome sequencing revealed that DE5 had a double-stranded DNA genome of 77, 305bp with 42.09% G+C content. A total of 126 open reading frames (ORFs) were identified, including functional genes related to phage integration, DNA replication and modification, transcriptional regulation, structural and packaging proteins, and host cell lysis. One phage integrase gene, one autotransporter adhesin gene, and one tRNA gene were predicted in the whole genome, and no genes associated with drug resistance were identified. The phage DE5 integrase contained 187 amino acids and belonged to the small serine recombinase family. BLASTn analysis revealed that phage DE5 had a high-sequence identity (96%) with E. coli phage SU10. Phylogenetic analysis showed that phage DE5 was a member of the genus Kuravirus. The whole genome sequencing of lysogenic phage DE5 enhanced our understanding of lysogenic phages and their therapeutic applications.

Emergence of IMP-8-Producing Comamonas thiooxydans Causing Urinary Tract Infection in China.

The emergence of carbapenem resistance (CR) caused by hydrolytic enzymes called carbapenemases has become a major concern worldwide. So far, CR genes have been widely detected in various bacteria. However, there is no report of CR gene harboring Comamonas thiooxydans. We first isolated a strain of an IMP-8-producing C. thiooxydans from a patient with urinary tract infection in China. Species identity was determined using MALDI-TOF MS analysis and carbapenemase-encoding genes were detected using PCR. The complete genomic sequence of C. thiooxydans was identified using Illumina Novaseq and Oxford Nanopore PromethION. Antimicrobial susceptibility analysis indicated that the C. thiooxydans strain ZDHYF418 was susceptible to imipenem, intermediate to meropenem, and was resistant to aztreonam, fluoroquinolones, and aminoglycosides. The blaIMP–8 gene was chromosomally located, and was part of a Tn402-like class 1 integron characterized by the following structure: DDE-type integrase/transposase/recombinase-tniB-tniQ-recombinase family protein-aac(6′)-Ib-cr-blaIMP–8-intI1. Phylogenetic analysis demonstrated that the closest relative of ZDHYF418 is C. thiooxydans QYY (accession number: CP053920.1). We detected 330 SNP differences between ZDHYF418 and C. thiooxydans QYY. Strain QYY was isolated from activated sludge in Jilin province, China in 2015. In summary, we isolated a strain of C. thiooxydans that is able to produce IMP-8 and a novel blaOXA. This is the first time that a CR gene has been identified in C. thiooxydans. The occurrence of the strain needs to be closely monitored.

SMART: recent updates, new developments and status in 2020.

SMART (Simple Modular Architecture Research Tool) is a web resource (https://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 9 contains manually curated models for more than 1300 protein domains, with a topical set of 68 new models added since our last update article (1). All the new models are for diverse recombinase families and subfamilies and as a set they provide a comprehensive overview of mobile element recombinases namely transposase, integrase, relaxase, resolvase, cas1 casposase and Xer like cellular recombinase. Further updates include the synchronization of the underlying protein databases with UniProt (2), Ensembl (3) and STRING (4), greatly increasing the total number of annotated domains and other protein features available in architecture analysis mode. Furthermore, SMART’s vector-based protein display engine has been extended and updated to use the latest web technologies and the domain architecture analysis components have been optimized to handle the increased number of protein features available.

A Rad51-independent pathway promotes single-strand template repair in gene editing.

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

Role of enterotoxigenic Escherichia coli prophage in spreading antibiotic resistance in a porcine-derived environment.

Enterotoxigenic Escherichia coli (ETEC) cause acute secretory diarrhoea in pigs, posing a great economic loss to the swine industry. This study analysed the prevalence and genetic characteristics of prophages from 132 ETEC isolates from symptomatic pigs to determine their potential for spreading antibiotic resistance. A total of 1105 potential prophages were identified, and the distribution of the genome size showed three 'overlapping' trends. Similarity matrix comparison showed that prophages correlated with the ETEC lineage distribution, and further identification of these prophages corroborated the lineage specificity. In total, 1206 antibiotic resistance genes (ARGs) of 52 different categories were identified in 132 ETEC strains; among these, 2.65% (32/1206) of ARGs were found to be carried by prophages. Analysis of flanking sequences showed that almost all the ARGs could be grouped into two types: 'blaTEM-1B ' and 'classic class 1 integron (IntI1)'. They co-occurred with a strictly conserved recombinase and transposon Tn3 family but with a difference: the 'blaTEM-1B type' prophages exhibited a classic Tn2 transposon structure with 100% sequence identity, whereas the 'IntI1 type' co-occurred with the TnAs2 transposon with only 84% sequence identity. These results imply that ARGs might be pervasive in natural bacterial populations through transmission by transposable bacteriophages.

Characterization of an archaeal recombinase paralog that exhibits novel anti-recombinase activity

The process of homologous recombination is heavily dependent on the RecA family of recombinases for repair of DNA double-strand breaks. These recombinases are responsible for identifying homologies and forming heteroduplex DNA between substrate ssDNA and dsDNA templates, activities that are modified by various accessory factors. In this work we describe the biochemical functions of the SsoRal2 recombinase paralog from the crenarchaeon Sulfolobus solfataricus. We found that the SsoRal2 protein is a DNA-independent ATPase that, unlike the other S. solfataricus paralogs, does not bind either ss- or dsDNA. Instead, SsoRal2 alters the ssDNA binding activity of the SsoRadA recombinase in conjunction with another paralog, SsoRal1. In the presence of SsoRal1, SsoRal2 has a modest effect on strand invasion but effectively abrogates strand exchange activity. Taken together, these results indicate that SsoRal2 assists in nucleoprotein filament modulation and control of strand exchange in S. solfataricus.

A soil bacterial catabolic pathway on the move: Transfer of nicotine catabolic genes between Arthrobacter genus megaplasmids and invasion by mobile elements

The 165,137 bp plasmid pAO1 of Paenarthrobacter nicotinovorans carries the genes of a nicotine catabolic pathway. The genes are organized into several gene modules responsible for the catabolism of L- and D-nicotine to nicotine blue, alpha-ketoglutarate and succinate. Various modules of these genes have been shown to be present in gram-positive (Gram?) soil bacteria. The presence of the identical pAO1 nic-genes on the 288,370 bp plasmid pZXY21 of Arthrobacter sp. ZXY2 (96 percent to 100 percent at the nucleotide level) permitted the identification of the limits of this DNA fragment. At the 5' end of the nic-genes are located the ORFs of two predicted integrases of the tyrosine recombinase family with conserved R, H, R and Y catalytic residues and that of a small transposase with a predicted leucine zipper motive. They are related to Tn554A, Tn554B and Tn554C of Staphylococcus aureus and suggest that the entire nic-genes DNA fragment represents a large catabolic transposon. Surprisingly the nic-genes on pZXY21 were found to be interspersed by mobile elements encoding transposases of various IS families. Insertion of these IS elements disrupts nicotine degradation and divide the nic-genes DNA into potentially new transposons. This finding may illustrate how nicotine catabolic genes can be mobilized and spread by horizontal gene transfer to other soil bacteria.

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance

Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.

Identification and Characterization of oriT and Two Mobilization Genes Required for Conjugative Transfer of Salmonella Genomic Island 1.

The integrative mobilizable elements of SGI1-family considerably contribute to the spread of resistance to critically important antibiotics among enteric bacteria. Even though many aspects of SGI1 mobilization by IncA and IncC plasmids have been explored, the basic transfer elements such as oriT and self-encoded mobilization proteins remain undiscovered. Here we describe the mobilization region of SGI1 that is well conserved throughout the family and carries the oriTSGI1 and two genes, mpsA and mpsB (originally annotated as S020 and S019, respectively) that are essential for the conjugative transfer of SGI1. OriTSGI1, which is located in the vicinity of the two mobilization genes proved to be a 125-bp GC-rich sequence with several important inverted repeat motifs. The mobilization proteins MpsA and MpsB are expressed from a bicistronic mRNA, although MpsB can be produced from its own mRNA as well. The protein structure predictions imply that MpsA belongs to the lambda tyrosine recombinase family, while MpsB resembles the N-terminal core DNA binding domains of these enzymes. The results suggest that MpsA may act as an atypical relaxase, which needs MpsB for SGI1 transfer. Although the helper plasmid-encoded relaxase proved not to be essential for SGI1 transfer, it appeared to be important to achieve the high transfer rate of the island observed with the IncA/IncC-SGI1 system.

The integrase of the Macrococcus caseolyticus resistance island mecD (McRImecD ) inserts DNA site-specifically into Staphylococcus and Bacillus chromosomes.

The methicillin resistance gene mecD has been recently identified on chromosomal islands in Macrococcus caseolyticus (McRImecD ). The 5' end of McRImecD carries an integrase (int) of the tyrosine recombinase family and two genes (intR and xis) encoding putative DNA-binding proteins. The islands are integrated site-specifically at the 3' end of the rpsI gene, a highly conserved locus in Gram-positive bacteria. Moreover, the rpsI gene of some Staphylococcus and Bacillus strains was found to be followed by a related integrase, raising the question of whether McRImecD could be transferred to these species. We used circular model elements carrying 5' end fragments of McRImecD -1 to demonstrate that the int enzyme and the attachment (att) site were sufficient to mediate site-specific DNA integration into the rpsI locus of Staphylococcus aureus, Staphylococcus pseudintermedius and Bacillus thuringiensis in vivo. Including xis in the model element stimulated both integrative and excisive recombination reactions and influenced the Int enzyme in att site selection. The intR gene functions as a negative regulator of int and xis. The int-xis genes of McRImecD -1 encode a site-specific recombination function that enables the acquisition of McRImecD in new hosts and the potential dissemination of broad-spectrum β-lactam resistance across genus barriers.

Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition.

IS607-family transposons are unusual because they do not have terminal inverted repeats or generate target site duplications. They encode two protein-coding genes, but only tnpA is required for transposition. Our X-ray structures confirm that TnpA is a member of the serine recombinase (SR) family, but the chemically-inactive quaternary structure of the dimer, along with the N-terminal location of the DNA binding domain, are different from other SRs. TnpA dimers from IS1535 cooperatively associate with multiple subterminal repeats, which together with additional nonspecific binding, form a nucleoprotein filament on one transposon end that efficiently captures a second unbound end to generate the paired-end complex (PEC). Formation of the PEC does not require a change in the dimeric structure of the catalytic domain, but remodeling of the C-terminal α-helical region is involved. We posit that the PEC recruits a chemically-active conformer of TnpA to the transposon end to initiate DNA chemistry.

Regulation of Hed1 and Rad54 binding during maturation of the meiosis-specific presynaptic complex.

Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double-strand break (DSB) repair, and the meiosis-specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis-specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real-time single-molecule imaging to probe how the Hed1- and Rad54-mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.

Determining the RAD51-DNA Nucleoprotein Filament Structure and Function by Cryo-Electron Microscopy.

Homologous recombination is a universal tool for DNA double-strand break and replication fork repair, and it is catalyzed by a highly conserved family of recombinases. In eukaryotes, Rad51 is the recombinase that catalyzes the pairing of homologous DNA molecules and the exchange of strands between the paired molecules. Rad51 assembles on single-stranded DNA (ssDNA) stemming from lesion processing to form a right-handed helical polymer that engages then samples double-stranded DNA (dsDNA) for homology. Upon matching with a homologous sequence, the Rad51-bound ssDNA invades the dsDNA, leading to the formation of a DNA joint with concomitant displacement of the strand of like polarity. The Rad51-DNA filaments are amenable to structural studies using cryo-electron microscopy (cryo-EM). In particular, recent technical breakthroughs in cryo-EM have made it possible to define the structure and function of human RAD51 at near-atomic resolution. In this chapter, we describe our cryo-EM approach to capture the human RAD51 filament structures in various stages of catalysis. The approach may also be useful for related recombinases and other helical assemblies.

Rad51 Interacts with Non-structural 3 Protein of Hepatitis C Virus and Regulates Viral Production.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV exploits host cellular factors for viral propagation. To investigate the cellular factors required for HCV propagation, we screened a siRNA library targeting human cell cycle genes using cell culture grown HCV-infected cells. In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of double-strand DNA breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was coprecipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 on the lipid raft. These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV.

Sequence imperfections and base triplet recognition by the Rad51/RecA family of recombinases

Homologous recombination plays key roles in double-strand break repair, rescue, and repair of stalled replication forks and meiosis. The broadly conserved Rad51/RecA family of recombinases catalyzes the DNA strand invasion reaction that takes place during homologous recombination. We have established single-stranded (ss)DNA curtain assays for measuring individual base triplet steps during the early stages of strand invasion. Here, we examined how base triplet stepping by RecA, Rad51, and Dmc1 is affected by DNA sequence imperfections, such as single and multiple mismatches, abasic sites, and single nucleotide insertions. Our work reveals features of base triplet stepping that are conserved among these three phylogenetic lineages of the Rad51/RecA family and also reveals lineage-specific behaviors reflecting properties that are unique to each recombinase. These findings suggest that Dmc1 is tolerant of single mismatches, multiple mismatches, and even abasic sites, whereas RecA and Rad51 are not. Interestingly, the presence of single nucleotide insertion abolishes recognition of an adjacent base triplet by all three recombinases. On the basis of these findings, we describe models for how sequence imperfections may affect base triplet recognition by Rad51/RecA family members, and we discuss how these models and our results may relate to the different biological roles of RecA, Rad51, and Dmc1.