RecA-mediated Homologous Recombination Research Articles

RecN spatially and temporally controls RecA-mediated repair of DNA double-strand breaks

RecN, a bacterial structural maintenance of chromosomes-like protein, plays an important role in maintaining genomic integrity by facilitating the repair of DNA double-strand breaks (DSBs). However, how RecN-dependent chromosome dynamics are integrated with DSB repair remains unclear. Here, we investigated the dynamics of RecN in response to DNA damage by inducing RecN from the PBAD promoter at different time points. We found that mitomycin C (MMC)-treated ΔrecN cells exhibited nucleoid fragmentation and reduced cell survival; however, when RecN was induced with arabinose in MMC-exposed ΔrecN cells, it increased a level of cell viability to similar extent as WT cells. Furthermore, in MMC-treated ΔrecN cells, arabinose-induced RecN colocalized with RecA in nucleoid gaps between fragmented nucleoids and restored normal nucleoid structures. These results suggest that the aberrant nucleoid structures observed in MMC-treated ΔrecN cells do not represent catastrophic chromosome disruption but rather an interruption of the RecA-mediated process. Thus, RecN can resume DSB repair by stimulating RecA-mediated homologous recombination, even when chromosome integrity is compromised. Our data demonstrate that RecA-mediated presynapsis and synapsis are spatiotemporally separable, wherein RecN is involved in facilitating both processes presumably by orchestrating the dynamics of both RecA and chromosomes, highlighting the essential role of RecN in the repair of DSBs.

An IS-mediated, RecA-dependent, bet-hedging strategy in Burkholderia thailandensis.

Adaptation to fluctuating environmental conditions is difficult to achieve. Phase variation mechanisms can overcome this difficulty by altering genomic architecture in a subset of individuals, creating a phenotypically heterogeneous population with subpopulations optimized to persist when conditions change, or are encountered, suddenly. We have identified a phase variation system in Burkholderia thailandensis that generates a genotypically and phenotypically heterogeneous population. Genetic analyses revealed that RecA-mediated homologous recombination between a pair of insertion sequence (IS) 2-like elements duplicates a 208.6 kb region of DNA that contains 157 coding sequences. RecA-mediated homologous recombination also resolves merodiploids, and hence copy number of the region is varied and dynamic within populations. We showed that the presence of two or more copies of the region is advantageous for growth in a biofilm, and a single copy is advantageous during planktonic growth. While IS elements are well known to contribute to evolution through gene inactivation, polar effects on downstream genes, and altering genomic architecture, we believe that this system represents a rare example of IS element-mediated evolution in which the IS elements provide homologous sequences for amplification of a chromosomal region that provides a selective advantage under specific growth conditions, thereby expanding the lifestyle repertoire of the species.

Modulation of RecFORQ- and RecA-Mediated Homologous Recombination in Escherichia coli by Isoforms of Translation Initiation Factor IF2.

Homologous recombination (HR) is critically important for chromosomal replication, as well as DNA damage repair in all life forms. In Escherichia coli, the process of HR comprises (i) two parallel presynaptic pathways that are mediated, respectively, by proteins RecB/C/D and RecF/O/R/Q; (ii) a synaptic step mediated by RecA that leads to generation of Holliday junctions (HJs); and (iii) postsynaptic steps mediated sequentially by HJ-acting proteins RuvA/B/C followed by proteins PriA/B/C of replication restart. Combined loss of RuvA/B/C and a DNA helicase UvrD is synthetically lethal, which is attributed to toxicity caused by accumulated HJs since viability in these double mutant strains is restored by removal of the presynaptic or synaptic proteins RecF/O/R/Q or RecA, respectively. Here we show that, as in ΔuvrD strains, ruv mutations confer synthetic lethality in cells deficient for transcription termination factor Rho, and that loss of RecFORQ presynaptic pathway proteins or of RecA suppresses this lethality. Furthermore, loss of IF2-1 (which is one of three isoforms [IF2-1, IF2-2, and IF2-3] of the essential translation initiation factor IF2 that are synthesized from three in-frame initiation codons in infB) also suppressed uvrD-ruv and rho-ruv lethalities, whereas deficiency of IF2-2 and IF2-3 exacerbated the synthetic defects. Our results suggest that Rho deficiency is associated with an increased frequency of HR that is mediated by the RecFORQ pathway along with RecA. They also lend support to earlier reports that IF2 isoforms participate in DNA transactions, and we propose that they do so by modulation of HR functions. IMPORTANCE The process of homologous recombination (HR) is important for maintenance of genome integrity in all cells. In Escherichia coli, the RecA protein is a critical participant in HR, which acts at a step common to and downstream of two HR pathways mediated by the RecBCD and RecFOR proteins, respectively. In this study, an isoform (IF2-1) of the translation initiation factor IF2 has been identified as a novel facilitator of RecA's function in vivo during HR.

Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination

Recombinases polymerize along single-stranded DNA (ssDNA) at the end of a broken DNA to form a helical nucleofilament with a periodicity of ∼18 bases. The filament catalyzes the search and checking for homologous sequences and promotes strand exchange with a donor duplex during homologous recombination (HR), the mechanism of which has remained mysterious since its discovery. Here, by inserting mismatched segments into donor duplexes and using single-molecule techniques to catch transient intermediates in HR, we found that, even though 3 base pairs (bp) is still the basic unit, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in ∼9-bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions ∼9 bp before the match-mismatch joint. The homology checking and the strand exchange are thus separated in space, with the strand exchange lagging behind. Our data suggest that the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA-RecA filament to check homology in ∼9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post-strand-exchanged duplex.

Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites

Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gram-negative bacteria. LPS is translocated to the OM by the LPS transport (Lpt) system. In the human pathogen Pseudomonas aeruginosa, the periplasmic Lpt component, LptH, is essential for LPS transport, planktonic and biofilm growth, OM stability and infectivity. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization. The relevance of these sites for LptH activity was verified through plasmid-mediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. Complementation and protein expression analyses provided evidence that all mutated sites are important for LptH activity in vivo. It was observed that the lptH conditional mutant overcomes the lethality of nonfunctional lptH variants through RecA-mediated homologous recombination between the wild-type lptH gene in the genome and mutated copies in the plasmid. Finally, biochemical assays on purified recombinant proteins showed that some LptH variants are indeed specifically impaired in oligomerization, while others appear to have defects in protein folding and/or stability.

Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology

DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks. However, the process of recombinational repair between short DNA regions of accidental similarity can lead to fatal genomic rearrangements. Previous studies have probed how effectively RecA discriminates against interactions involving a short similar sequence that is embedded in otherwise dissimilar sequences but have not yielded fully conclusive results. Here, we present results of in vitro experiments with fluorescent probes strategically located on the interacting DNA fragments used for recombination. Our findings suggest that DNA synthesis increases the stability of the recombination products. Fluorescence measurements can also probe the homology dependence of the extension of invading DNA strands in D-loops formed by RecA-mediated strand exchange. We examined the slow extension of the invading strand in a D-loop by DNA polymerase (Pol) IV and the more rapid extension by DNA polymerase LF-Bsu. We found that when DNA Pol IV extends the invading strand in a D-loop formed by RecA-mediated strand exchange, the extension afforded by 82 bp of homology is significantly longer than the extension on 50 bp of homology. In contrast, the extension of the invading strand in D-loops by DNA LF-Bsu Pol is similar for intermediates with ≥50 bp of homology. These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase.

Gibson Assembly facilitates bacterial allelic exchange mutagenesis

Allelic exchange mutagenesis that relies on RecA-mediated homologous recombination up- and downstream from the targeted gene is a generalizable method of site-specific bacterial gene knock-out and knock-in. However, generation of a mutagenic DNA construct (alternative allele flanked by regions surrounding the gene target) and subsequent mutant selection are laborious procedures. Here we demonstrate allelic exchange knock-out facilitated by Gibson Assembly in Streptococcus iniae. Gibson Assembly allows rapid construction of a large allelic exchange cassette simultaneous with cloning, as well as rapid reconstruction of complete recombinant vector sequence when required. Additionally, we show that during two-step mutant selection, absence of recombination at one of the homologous regions (single cross-over) might be rapidly detected by colony PCR of meroploid clones and resolved by extension/shifting of corresponding sequence in DNA construct. The combination of Gibson Assembly for mutagenic DNA construction/redesign with colony PCR screening of meroploids to detect recombination at both sides of the exchange target may significantly accelerate generation of chromosomal mutants in a wide range of bacterial taxa.

Cooperative RecA clustering: the key to efficient homology searching

The mechanism by which pre-synaptic RecA nucleoprotein filaments efficiently locate sequence homology across genomic DNA remains unclear. Here, using atomic force microscopy, we directly investigate the intermediates of the RecA-mediated homologous recombination process and find it to be highly cooperative, involving multiple phases. Initially, the process is dominated by a rapid ‘association’ phase, where multiple filaments interact on the same dsDNA simultaneously. This cooperative nature is reconciled by the observation of localized dense clusters of pre-synaptic filaments interacting with the observed dsDNA molecules. This confinement of reactive species within the vicinity of the dsDNA, is likely to play an important role in ensuring that a high interaction rate between the nucleoprotein filaments and the dsDNA can be achieved. This is followed by a slower ‘resolution’ phase, where the synaptic joints either locate sequence homology and progress to a post-synaptic joint, or dissociate from the dsDNA. Surprisingly, the number of simultaneous synaptic joints decreases rapidly after saturation of the dsDNA population, suggesting a reduction in interaction activity of the RecA filaments. We find that the time-scale of this decay is in line with the time-scale of the dispersion of the RecA filament clusters, further emphasising the important role this cooperative phenomena may play in the RecA-facilitated homology search.

Iterative homology checking and non-uniform stepping during RecA-mediated strand exchange

Recombinase-mediated homologous recombination (HR) in which strands are exchanged between two similar or identical DNA molecules is essential for maintaining genome fidelity and generating genetic diversity. It is believed that HR comprises two distinct stages: an initial alignment with stringent homology checking followed by stepwise heteroduplex expansion. If and how homology checking takes place during heteroduplex expansion, however, remains unknown. In addition, the number of base pairs (bp) involved in each step is still under debate. By using single-molecule approaches to catch transient intermediates in RecA-mediated HR with different degrees of homology, we show that (i) the expansion proceeds with step sizes of multiples of 3 bp, (ii) the step sizes follow wide distributions that are similar to that of initial alignment lengths, and (iii) each distribution can be divided into a short-scale and a long-scale part irrespective of the degree of homology. Our results suggest an iterative mechanism of strand exchange in which ssDNA-RecA filament interrogates double-stranded DNA using a short tract (6–15 bp) for quick checking and a long tract (>18 bp) for stringent sequence comparison. The present work provides novel insights into the physical and structural bases of DNA recombination.

A defect in homologous recombination leads to increased translesion synthesis in E. coli.

DNA damage tolerance pathways allow cells to duplicate their genomes despite the presence of replication blocking lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS) and homology directed gap repair (HDGR). TLS pathways involve specialized DNA polymerases that are able to synthesize past DNA lesions with an intrinsic risk of causing point mutations. In contrast, HDGR pathways are essentially error-free as they rely on the recovery of missing information from the sister chromatid by RecA-mediated homologous recombination. We have investigated the genetic control of pathway choice between TLS and HDGR in vivo in Escherichia coli. In a strain with wild type RecA activity, the extent of TLS across replication blocking lesions is generally low while HDGR is used extensively. Interestingly, recA alleles that are partially impaired in D-loop formation confer a decrease in HDGR and a concomitant increase in TLS. Thus, partial defect of RecA's capacity to invade the homologous sister chromatid increases the lifetime of the ssDNA.RecA filament, i.e. the ‘SOS signal’. This increase favors TLS by increasing both the TLS polymerase concentration and the lifetime of the TLS substrate, before it becomes sequestered by homologous recombination. In conclusion, the pathway choice between error-prone TLS and error-free HDGR is controlled by the efficiency of homologous recombination.

Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome

Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.

The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA

The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation (<1%) of cells in a stationary-phase culture. RcGTA particles deliver this DNA to surrounding R. capsulatus cells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the lexA gene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in the R. capsulatus genome sequence identified a number of putative binding sites located 5' of typical SOS response coding sequences and also 5' of the RcGTA regulatory gene cckA, which encodes a hybrid histidine kinase homolog. Expression of cckA was increased >5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ΔcckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like genetic exchange carried out by the R. capsulatus gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA gene expression differs radically.

Combination of magnetic tweezers with DNA hairpin as a potential approach to the study of RecA-mediated homologous recombination

Homologous recombination（HR) is essential for maintaining the genome fidelity and generating genetic diversity. As a prototypical member of the recombinases, RecA from Escherichia coli has been extensively studied by using single-molecule FRET（smFRET), magnetic tweezers, optical tweezers, etc. However, these methods cannot meet the needs of wide-ranged observations nor high spatial resolution at the same time. For sequence comparison, the average base-to-base distance of the homologous dsDNA will be stretched from 0.34 nm to 0.51 nm. The increment for per base pair is 0.17 nm, which is far beyond the spatial resolution of magnetic tweezers so that it cannot be directly measured. As a high-resolution technique, the smFRET enables us to observe more details of reactions. However, its valid measuring distance is 3-8 nm, which limits the observation range. Here, we propose an approach by combining magnetic tweezers with DNA hairpin, which may solve the problem effectively in the study of HR. In this paper, one end of the DNA molecule with a 270 bp hairpin is immobilized onto the surface of the flow cell, while a magnetic bead is attached to the other end. An external magnetic force is applied to the magnetic bead by placing a permanent magnet above the flow cell. The first 90 bp（from the junction of the hairpin) of the hairpin is homologous to the ssDNA within the ssDNA-RecA filament. Thus, the filament searches for homology along the hairpin, and incorporates into the homologous segment for strand exchange. After that, the displaced strand can be opened by pulling at a force of ~7 pN, and each opened base pair results in a 0.82 nm increase in DNA extension. By using this approach, we show that 1) RecA-mediated strand exchange proceeds in a stepwise manner and the average speed is ~7.6 nt/s, which is in accordance with previous result; 2) the dynamic interaction between the second DNA-binding site（SBS) and the displaced strand can be observed in real-time, and the binding force is calculated accurately through the x-dimensional fluctuations; 3) the processes of strand-exchange in different directions can be observed, and the directions are distinguishable through the reaction patterns. The results suggest that the combination of magnetic tweezers with DNA hairpin is a potential approach to the study of RecA or other recombinases. Therefore, our design can be an important single-molecule approach to the research of HR mechanism.

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.

An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments.

BackgroundThe Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium.ResultsWe constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination.ConclusionsWe developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1425-4) contains supplementary material, which is available to authorized users.

Freeing Pseudomonas putida KT2440 of its proviral load strengthens endurance to environmental stresses.

2.6% of the genome of the soil bacterium Pseudomonas putida KT2440 encodes phage-related functions, but the burden of such opportunistic DNA on the host physiology is unknown. Each of the four apparently complete prophages borne by this strain was tested for stability, spontaneous excision and ability to cause lysis under various stressing conditions. While prophages P3 (PP2266-PP2297) and P4 (PP1532-1584) were discharged from the genome at a detectable rate, their induction failed otherwise to yield infective viruses. Isogenic P. putida KT2440 derivatives bearing single and multiple deletions of each of the prophages were then subjected to thorough phenotypic analyses, which generally associated the loss of proviral DNA with an increase of physiological vigour. The most conspicuous benefit acquired by prophage-less cells was a remarkable improvement in tolerance to UV light and other insults to DNA. This was not accompanied, however, with an upgrade of recA-mediated homologous recombination. The range of tolerance to DNA damage gained by the prophage-free strain was equivalent to the UV resistance endowed by the TOL plasmid pWW0 to the wild-type bacterium. While the P. putida's prophages are therefore genuinely parasitic, their detrimental effects can be offset by acquisition of compensatory traits through horizontal gene transfer.

Single-Molecule Fluorescence Imaging of Reco Localization and Dynamics in Bacillus Subtilis

In all organisms, the high fidelity of DNA replication is essential for maintenance of chromosome integrity. DNA damage can be caused by polymerase errors or by external factors (e.g., X-rays or mutagenic chemicals). Thus, the cell has evolved a number of repair mechanisms to respond to different types of damage. In B. subtilis, repair of double-strand breaks (DSBs) in the DNA occurs through RecA-mediated homologous recombination. The mechanism by which RecA finds DSBs in vivo is not well described, but is believed to involve the proteins RecF, RecO, and RecR. This role for RecO in DSB repair in B. subtilis is distinct from its role in E. coli but analogous to that of Rad52 in eukaryotes, making B. subtilis an excellent model system for studying cellular response to DNA damage. Previously, bulk fluorescence studies have shown that RecO forms foci after the induction of double-strand breaks. However, RecO in undamaged cells can only be visualized when overexpressed, leaving questions about its true localization at wild type expression levels. Here, we have created cells in which PAmCherry-RecO is natively expressed from the RecO promoter as the only RecO source. We use single-molecule fluorescence microscopy in live B. subtilis to show that RecO rapidly forms foci following treatment with the DNA damaging agent phleomycin, but is diffuse throughout the cell under non-damaging conditions. This result suggests that, unlike several other proteins involved in DNA repair, RecO is not associated with the replisome prior to DSB recognition. Future work will examine the previously reported role of single-strand binding protein (SSB) in recruitment of RecO to DSBs.

Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis

BackgroundThe Bacillus subtilis genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genome of B. subtilis functions as a vector. The BGM vector system has several attractive properties, such as a large cloning capacity of over 3 Mb, stable propagation of cloned DNA and various modification strategies using RecA-mediated homologous recombination. However, genetic modifications using the BGM vector system have not been fully established, and this system has not been applied to transgenesis. In this study, we developed important additions to the genetic modification methods of the BGM vector system. To explore the potential of the BGM vector, we focused on the fish-like odorant receptor (class I OR) gene family, which consists of 158 genes and forms a single gene cluster. Although a cis-acting locus control region is expected to regulate transcription, this has not yet been determined experimentally.ResultsUsing two contiguous bacterial artificial chromosome clones containing several class I OR genes, we constructed two transgenes in the BGM vector by inserting a reporter gene cassette into one class I OR gene. Because they were oriented in opposite directions, we performed an inversion modification to align their orientation and then fused them to enlarge the genomic structure. DNA sequencing revealed that no mutations occurred during gene manipulations with the BGM vector. We further demonstrated that the modified, reconstructed genomic DNA fragments could be used to generate transgenic mice. Transgenic mice carrying the enlarged transgene recapitulated the expression and axonal projection patterns of the target class I OR gene in the main olfactory system.ConclusionWe offer a complete genetic modification method for the BGM vector system, including insertion, deletion, inversion and fusion, to engineer genomic DNA fragments without any trace of modifications. In addition, we demonstrate that this system can be used for mouse transgenesis. Thus, the BGM vector system can be an alternative platform for engineering large DNA fragments in addition to conventional systems such as bacterial and yeast artificial chromosomes. Using this system, we provide the first experimental evidence of a cis-acting element for a class I OR gene.

The role of Deinococcus radiodurans RecFOR proteins in homologous recombination

Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed.

Lactococcal phage p2 ORF35‐Sak3 is an ATPase involved in DNA recombination and AbiK mechanism

Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35(p2), also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35(p2) form a hexameric ring-like structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35(p2) interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35(p2) is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35(p2) is a novel SSAP stimulating homologous recombination.