Mechanisms of homology-facilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri

Intra- and interspecific natural transformation has been observed in many prokaryotic species and is considered a fundamental mechanism for the generation of genetic variation. Recently, it has been described in detail how, in transformable Acinetobacter BD413 and Streptococcus pneumoniae, long stretches of nucleotides lacking homology were integrated into recipient genomes when they were linked on one side to a small piece of DNA with homology to resident DNA serving as a recA-dependent recombination anchor. Now, such homology-facilitated illegitimate recombination (HFIR) has also been detected in transformable Pseudomonas stutzeri. However, analysis of the recombinants revealed qualitative and quantitative differences in their generation compared with that in Acinetobacter BD413. In P. stutzeri, foreign DNA with an anchor sequence was integrated 105- to 106-fold less frequently than fully homologous DNA, but still at least 200-fold more frequently than without the anchor. The anchor sequence could be as small as 311 bp. Remarkably, in 98% of the events, the 3' end was integrated within the homologous anchor, whereas the 5' end underwent illegitimate fusion. Moreover, about one-third of the illegitimate fusion sites shared no or only a single identical basepair in foreign and resident DNA. The other fusions occurred within microhomologies of up to 6 bp with a higher GC content on average than the interacting nucleotide sequences. Foreign DNA of 69-1903 bp was integrated, and resident DNA of 22-2345 bp was lost. In a recA mutant, HFIR was not detectable. The findings suggest that genomic acquisition of foreign DNA by HFIR during transformation occurs widely in prokaryotes, but that details of the required recombination and strand fusion mechanisms may differ between organisms from different genera.     

Mechanism of illegitimate recombination: common sites for recombination and cleavage mediated by E. coli DNA gyrase

Summary Illegitimate recombination dependent on DNA gyrase in a cell-free system has previously been described. We have now mapped DNA gyrase cleavage sites in the vicinity of known recombination sites in pBR322. Among five recombination sites examined, three were found to coincide with a DNA gyrase cleavage site. This result suggests that the cleavage of DNA by DNA gyrase has a central role in the recombination process.     

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe

Molecular Genetics and Genomics - In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate...     

Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype.

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we isolated temperature-sensitive gyrA mutants that confer spontaneous illegitimate recombination and spontaneous induction of lambda prophage at higher frequencies than that in the wild-type. After reconstruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyrAI203V+gyrAI205V, show the same properties as those described above. With respect to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of recombination junctions of lambdabio transducing phages formed spontaneously in these mutants showed that the parental E. coli bio and lambda recombination sites have a homologous sequence of only 0. 7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysis of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrAL488P mutations contain amino acid substitutions of Leu492-->Pro and Leu488-->Pro, respectively, which correspond to the alpha18 helix in the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gyrAI205V mutations contain Ile203-->Val and Ile205-->Val, respectively, which correspond to the alpha10' helix, also in the breakage-reunion domain of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 protein that contains the L492P mutation has an apparently normal supercoiling activity, but it also produces a small amount of linear DNA in the absence of DNA gyrase inhibitor during the supercoiling reaction, suggesting that the mutant DNA gyrase may have a defect at the step of religation or a defect in the subunit interaction. These results suggest that the recombination is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two alpha helices, alpha10' and alpha18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA.     

Molecular analysis of DNA junctions produced by illegitimate recombination in human cells.

In a human HeLa derived-cell line carrying permanently a single integrated copy of an SV40 shuttle vector, the transient expression of the SV40 T-antigen led to the production of heterogeneous populations of circular DNA molecules which retained both integrated vector and its surrounding cellular sequences. Comparison between the integrated copy and the linear maps of 80 different plasmids rescued in bacteria suggested that the formation of circular DNA was the result of bidirectional replication from the SV40 origin of replication followed by a single intramolecular joining leading to the cyclization of the replicated molecules. Sequence analysis of 45 recombinational junctions demonstrated that the cyclization occurred via illegitimate recombination process which did not require preferential nucleotide sequence at the joining sites. However, extensive characterization of recombination junctions revealed that the sequences involved in the recombination at each side of the SV40 origin of replication were not randomly distributed, suggesting the presence of regions which were more prone to be involved in the illegitimate recombination process in human cells. Search of common features usually implied in illegitimate recombination in mammalian cells revealed some association of these regions with palindromes, A + T-rich DNA segments, alternating purine/pyrimidine sequences and Alu family repeats.     

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1 ⁺ gene was present in the introduced leu1::ura4 ⁺ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of nonhomologous to homologous recombination events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites. Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.     

Processing of DNA prior to illegitimate recombination in mouse cells.

In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 5'-->3' exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 3'-->5' exonuclease processing also occurs.     

Molecular mechanisms of exon shuffling: illegitimate recombination

Illegitimate recombination (IR) is a process that takes place far more often than homologous recombination and is characterized by the recombination between non-homologous or short homologous sequences. The consequences of IR frequently emerge after the introduction of DNA in cell lines because it more frequently integrates in non-homologous than in homologous regions of the host genome. As a result, unexpected truncated or elongated products may be found. By not discarding those products as transfection artifacts, but by studying how they are generated, it might elucidate a possible molecular mechanism of IR. Here we review the current literature describing different mechanisms by which non-homologous DNA recombination can be induced.     

DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics

Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast- and slow-growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic‘toolbox’for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.     

Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein-DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.     

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I.

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

Involvement of DNA replication in phage lambda Red-mediated homologous recombination

Crosses between a non-replicating linear bacteriophage lambda chromosome and a replicating plasmid bearing a short cloned segment of lambda DNA were monitored by extracting DNA from infected cells, and analysing it via restriction endonuclease digestion and Southern blots. Recombinant formation resulting from the action of the Red homologous recombination system, observed directly in this way, was found to be fast, efficient, independent of the bacterial recA function and highly dependent upon replication of the target plasmid. These features of the experimental system faithfully model Red-mediated recombination in a lytically infected cell in which phage DNA replication is occurring. Neither of the previously established mechanisms by which the Red system can operate – strand annealing or strand invasion – accounts well for these findings. A third mechanism, replisome invasion, involving replication directly in the recombination mechanism, is invoked as an alternative.     

Genomic and proteomic characterization of the broad-host-range Salmonella phage PVP-SE1: creation of a new phage genus

(Bacterio)phage PVP-SE1, isolated from a German wastewater plant, presents a high potential value as a biocontrol agent and as a diagnostic tool, even compared to the well-studied typing phage Felix 01, due to its broad lytic spectrum against different Salmonella strains. Sequence analysis of its genome (145,964 bp) shows it to be terminally redundant and circularly permuted. Its G+C content, 45.6 mol%, is lower than that of its hosts (50 to 54 mol%). We found a total of 244 open reading frames (ORFs), representing 91.6% of the coding capacity of the genome. Approximately 46% of encoded proteins are unique to this phage, and 22.1% of the proteins could be functionally assigned. This myovirus encodes a large number of tRNAs (n=24), reflecting its lytic capacity and evolution through different hosts. Tandem mass spectrometric analysis using electron spray ionization revealed 25 structural proteins as part of the mature phage particle. The genome sequence was found to share homology with 140 proteins of the Escherichia coli bacteriophage rV5. Both phages are unrelated to any other known virus, which suggests that an "rV5-like virus" genus should be created within the Myoviridae to contain these two phages.     

The stereostructure of knots and catenanes produced by phage lambda integrative recombination: implications for mechanism and DNA structure

We studied the mechanism of recombination by determining the structure of the products of the phage lambda Int system. Electron microscopy of RecA-coated products revealed only knots and catenanes containing a regular right-handed spiral structure. The structure and distribution of products establish that the recombination sites pair by essentially random collision, rather than by tracking. However, the distribution also indicates that the binding of the enzyme must introduce nonrandom components into the reaction and stabilize at least two additional supercoils that become links in the product. Moreover, the regularity of the structures indicates that the strand exchange is accomplished in a very simple way, introducing only a single link into the product. All other links result from the direct conversion of substrate supercoils into knot and catenane links. These supercoils must be in a right-handed, braided form, rather than solenoidally wound as in nucleosomes.     

Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage

RSΒ1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The RSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of RSB1 were demonstrated.     

Effect of the DNA topoisomerase II inhibitor VP-16 on illegitimate recombination in yeast chromosomes

Etoposide and teniposide, derivatives of podophyllotoxin, are inhibitors of DNA topoisomerase II and are potent anticancer agents. An adverse effect linked to the use of these drugs is the development of acute myeloid leukemia, a disorder usually associated with chromosomal translocation. To examine podophyllotoxin-induced DNA rearrangement, we developed an assay system to measure illegitimate recombination in Saccharomyces cerevisiae chromosomes. This approach uses juxtaposed CAN1-CYH2 negative selection markers that are introduced into the LEU2 locus, which is located on chromosome III, in a yeast strain carrying the mutated can1 and cyh2 genes. Upon formation of a deletion over the active CAN1-CYH2 genes, a cell becomes resistant to both canavanine and cycloheximide. To introduce drugs into the cell, we used a yeast strain carrying an ISE2 mutation, thereby making the cell drug-permeable. Here we show that treatment of cells with etoposide (VP-16) increases the rate of illegitimate recombination in yeast, indicating that VP-16 stimulates DNA topoisomerase-mediated illegitimate recombination. Structural analysis of the resulting recombinants indicate that most are formed by deletion mutations on chromosome III, which take place between short homologous regions of DNA. We propose a model for illegitimate recombination, in which VP-16 facilitates formation of a cleavable complex between DNA topoisomerase II and DNA, thus promoting DNA double-strand breakage with the resulting DNA ends joined by a non-homologous mechanism.     

Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli

We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism.     

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.