Importance of illegitimate recombination and transposition in IS30-associated excision events.

In the present study we report on the excision of IS30 elements and IS30-derived composite transposons. Frequent loss of IS30 was observed during dissolution of dimeric IS30 structures, containing IR-IR junctions, leading to resealed donor molecules. In contrast, unambiguous transpositional excision resulting in resealed remainder products could not be identified in the case of a monomeric element. The bias in the excision of monomeric and dimeric IS30 structures indicates a difference in the molecular mechanism of transposition of IS30 monomers and dimers. Sequence data on the rarely detected plasmids missing full IS or Tn copies rather suggest that all products were derived from illegitimate recombination. The reaction occurred between short homologies and was independent of the transposase activity. Similar IS30 excision events accompanied by multiple plasmid or genome rearrangements were detected in Pseudomonas putida and Rhizobium meliloti, yielding stable replicons that retained the selective marker gene of the transposon. We provide evidence that both transposition and illegitimate recombination can contribute to the stabilization of replicons through the elimination of IS elements, which emphasizes the evolutionary significance of these events.     

IS2-IS2 and IS3-IS3 relative recombination frequencies in F integration

The relative integrative recombination frequencies of the F plasmid IS2 and IS3 elements were determined at two Escherichia coli K-12 chromosomal sites by hybridization analysis of Hfr DNA. The sequence organizations of the independent Hfr strains formed by F integration atα₃β₃ indicate that the relative recombinational frequencies at the two F plasmid IS3 elementsα₁β₁ and α₂β₂ are not significantly different. A comparison of the relative recombinational frequencies of the IS2 and IS3 elements of F was provided by analysis of DNA from Hfr strains having F integrated betweenlacandproC (i.e., at the IS2 or at the IS3 element (α₅β₅) located in this region). No instances of F integration atα₅β₅ were detected, indicating that integrative recombination at IS2 is significantly more frequent than at IS3 in this chromosomal region.     

Chromatin loops,illegitimate recombination,and genome evolution

Chromosomal rearrangements frequently occur at specific places (“hot spots”) in the genome. These recombination hot spots are usually separated by 50–100 kb regions of DNA that are rarely involved in rearrangements. It is quite likely that there is a correlation between the above‐mentioned distances and the average size of DNA loops fixed at the nuclear matrix. Recent studies have demonstrated that DNA loop anchorage regions can be fairly long and can harbor DNA recombination hot spots. We previously proposed that chromosomal DNA loops may constitute the basic units of genome organization in higher eukaryotes. In this review, we consider recombination between DNA loop anchorage regions as a possible source of genome evolution.     

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...     

Origination of an X-linked testes chimeric gene by illegitimate recombination in Drosophila

The formation of chimeric gene structures provides important routes by which novel proteins and functions are introduced into genomes. Signatures of these events have been identified in organisms from wide phylogenic distributions. However, the ability to characterize the early phases of these evolutionary processes has been difficult due to the ancient age of the genes or to the limitations of strictly computational approaches. While examples involving retrotransposition exist, our understanding of chimeric genes originating via illegitimate recombination is limited to speculations based on ancient genes or transfection experiments. Here we report a case of a young chimeric gene that has originated by illegitimate recombination in Drosophila. This gene was created within the last 2–3 million years, prior to the speciation of Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana. The duplication, which involved the Bällchen gene on Chromosome 3R, was partial, removing substantial 3′ coding sequence. Subsequent to the duplication onto the X chromosome, intergenic sequence was recruited into the protein-coding region creating a chimeric peptide with ~ 33 new amino acid residues. In addition, a novel intron-containing 5′ UTR and novel 3′ UTR evolved. We further found that this new X-linked gene has evolved testes-specific expression. Following speciation of the D. simulans complex, this novel gene evolved lineage-specifically with evidence for positive selection acting along the D. simulans branch.     

Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.     

Structural variability of Tvv1 grapevine retrotransposons can be caused by illegitimate recombination

Structural variability of Tvv1, a grapevine retrotransposon Ty1 copia-like family, was investigated within the grape genome and the canonical sequence of Tvv1 determined. Then, two remarkable elements, Tvv1-Δ3001 and Tvv1-Δ3640, which had suffered large deletions 3,001 bp and 3,460 bp in length of their coding sequences were compared to the canonical copy. In both deleted elements, the deletion breakpoint was characterized by a stretch 13 bp-long in Tvv1-Δ3001 and 11 bp-long in Tvv1-Δ3640 found duplicated in the canonical copy at each bound of the deleted regions. Tvv1-Δ3001 and Tvv1-Δ3460 were both shown to be unique copies fixed at a single locus in the grapevine genome. Their presence was very variable in a set of 58 varieties and wild vines. These elements have most likely been dispersed through natural intermixing after their initial insertion whose chronology was estimated. The model that we propose to explain the structure of Tvv1-Δ3001 and Tvv1-Δ3640, implies illegitimate recombination involving template switching between two RNA molecules co-packaged in the VLP prior to the integration of the deleted daughter copy into the host genome.     

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.     

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.     

IS2-43 and IS2-44: New alleles of the insertion sequence IS2 which have promoter activity

Summary The sequence of two new IS2 alleles with promoter activity (IS2-43 and IS2-44) is reported. The alleles are identical and are formed by a 17 bp tandem duplication in an AT-rich region of IS2. This created a new RNA polymerase binding site. A mutation was found that increased the frequency of formation of these 17 bp duplications but not of another class of duplications, the mini-insertions. This suggested that the mechanisms of formation of the two classes of duplications are different.     

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.     

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.     

Suppression of gamma ray-induced illegitimate recombination in Escherichia coli by the DNA-binding protein H-NS

To study the mechanism of gamma-ray-induced illegitimate recombination, we examined the formation of lambdabio transducing phage in Escherichia coli after gamma-ray irradiation. We show that gamma-ray irradiation enhances the formation of lambdabio transducing phage during prophage induction. Moreover, an hns mutation synergistically enhanced the incidence of lambda-ray-induced illegitimate recombination. Next we determined the sequences at the recombination junctions of the lambdabio transducing phages induced by gamma-ray irradiation. Most of the recombination sites coincided with known hotspots. Among them, hotspot I accounted for 67% and 77% of gamma-ray-induced lambdabio transducing phages in the wild type and the hns mutant, respectively. Therefore, the recombination sites appear to occur mostly at hotspot I or at other hotspots, but rarely at non-hotspot sites. These results suggest that types of DNA damage other than the double-strand breaks induced at random sites are mainly responsible for the introduction of the site-specific or region-specific DNA double strand breaks that lead to recombination at the hotspots. The results also showed that the recombination events took place between DNA sequences possessing short stretches of homology. H-NS protein, which binds to curved DNA, suppresses illegitimate recombination in the presence and absence of gamma-ray irradiation. Models for gamma-ray-induced illegitimate recombination are discussed.     

Ionizing radiation and restriction enzymes induce microhomology-mediated illegitimate recombination in Saccharomyces cerevisiae

DNA double-strand breaks can be repaired by illegitimate recombination without extended sequence homology. A distinct mechanism namely microhomology-mediated recombination occurs between a few basepairs of homology that is associated with deletions. Ionizing radiation and restriction enzymes have been shown to increase the frequency of nonhomologous integration in yeast. However, the mechanism of such enhanced recombination events is not known. Here, we report that both ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated integration. Irradiated yeast cells displayed 77% microhomology-mediated integration, compared to 27% in unirradiated cells. Radiation-induced integration exhibited lack of deletions at genomic insertion sites, implying that such events are likely to occur at undamaged sites. Restriction enzymes also enhanced integration events at random non-restriction sites via microhomology-mediated recombination. Furthermore, generation of a site-specific I-SceI-mediated double-strand break induces microhomology-mediated integration randomly throughout the genome. Taken together, these results suggest that double-strand breaks induce a genome-wide microhomology-mediated illegitimate recombination pathway that facilitates integration probably in trans at non-targeted sites and might be involved in generation of large deletions and other genomic rearrangements.     

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.     

Sequence of the junction in adenovirus 2-SV40 hybrids: examples of illegitimate recombination.

The nucleotide sequences of six Ad2-SV40 junctions from three Ad2-SV40 hybrid viruses (Ad2++HEY, Ad2++LEY and Ad2+D1) were determined. Comparison of parental adenovirus 2 and SV40 DNA sequences with the sequence at the Ad2-SV40 junctions revealed that 5 out of 6 junctions are abrupt transitions from Ad2 to SV40 DNA, and in one case (Ad2++LEY, right junction) there is an additional nucleotide at the junction, which cannot be ascribed to either DNA. Ad2++HEY and Ad2+D1 right junctions are identical and Ad2++LEY and Ad2+ND4 left junctions are identical, a result that strongly suggests these Ad2-SV40 hybrids arose by recombination between the linear Ad2 DNA and circular SV40 DNA, followed by recombination between Ad2 DNA and SV40 DNA present in the Ad2-SV40 hybrid DNA. The unambiguous transition of Ad2 DNA into SV40 DNA at the junction sites is an example of recombination events which have apparently occurred without any homology at the recombination site.     

Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.