Sequential strand exchange by XerC and XerD during site-specific recombination at dif

Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges.     

Effects of adeno-associated virus DNA hairpin structure on recombination

Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.     

Intermolecular recombination of human c-myb proto-oncogene coding sequences

We have previously reported evidence suggesting that intermolecular recombination events are involved in the tissue specific expression of the c-myb proto-oncogene in chicken. We show in this paper that recombined c-myb mRNA species are also expressed in human thymic cells, therefore indicating that intermolecular recombination of coding sequences occurs in higher eucaryotes.     

Non-equivalent interactions between amino-terminal domains of neighboring lambda integrase protomers direct Holliday junction resolution

The bacteriophage lambda site-specific recombinase (Int), in contrast to other family members such as Cre and Flp, has an amino-terminal domain that binds "arm-type" DNA sequences different and distant from those involved in strand exchange. This defining feature of the heterobivalent recombinases confers a directionality and regulation that is unique among all recombination pathways. We show that the amino-terminal domain is not a simple "accessory" element, as originally thought, but rather is incorporated into the core of the recombination mechanism, where it is well positioned to exert its profound effects. The results reveal an unexpected pattern of intermolecular interactions between the amino-terminal domain of one protomer and the linker region of its neighbor within the tetrameric Int complex and provide insights into those features distinguishing an "active" from an "inactive" pair of Ints during Holliday junction resolution.     

Nonideal Statistics and Positive Correlation in Phage Recombination: Studies with λ Tandem Duplication Phages

The question of nonideality in phage recombination, that is, the extent to which recombinant frequencies differ from those expected from the proportions of the two parental types in the mass culture, was addressed by experiments with λ tandem duplication phages. Isolation and genotypic analysis of triplication-phage progeny, all of which must be the result of intermolecular recombination, yielded a value of about 0.5 for the nonideality parameter h, i.e., the frequency of unlike-parent matings was only about ½ the "ideal" value. This value was independent of multiplicity and about the same for the Rec or Red recombination systems. Similar analysis of single-copy phage progeny yielded estimates of k, the ratio of intramolecular to intermolecular recombination of about for the Rec system; no intramolecular events were detected in Red-mediated crosses. Consideration of known nonideality factors (finite input, limited number of intracellular sites for phage growth) suggests that the observed h values correspond to intracellular mixing efficiencies of 55 to 100%, depending on the number of intracellular phage growth sites assumed. Analysis of long-range positive correlation (negative interference) indicates that statistical effects caused unlike-parent double crossovers to be three to four times as frequent as an independent-event calculation would predict. In addition, Rec-mediated crosses showed a 1.3-fold positive correlation for unlike-parent crossovers (in a second interval) among the progeny of like-parent recombinations.     

Intermolecular V(D)J recombination

V(D)J recombination plays a prominent role in the generation of the antigen receptor repertoires of B and T lymphocytes. It is also likely to be involved in the formation of chromosomal translocations, some of which may result from interchromosomal recombination. We have investigated the potential of the V(D)J recombination machinery to perform intermolecular recombination between two plasmids, either unlinked or linked by catenation. In either case, recombination occurs in trans to yield signal and coding joints, and the results do not support the existence of a mechanistic block to the formation of coding joints in trans. Instead, we observe that linearization of the substrate, which does not alter the cis or trans status of the recombination signals, causes a specific and dramatic reduction in coding joint formation. This unexpected result leads us to propose a "release and recapture" model for V(D)J recombination in which coding ends are frequently released from the postcleavage complex and the efficiency of coding joint formation is influenced by the efficiency with which such ends are recaptured by the complex. This implies the existence of mechanisms, operative during recombination of chromosomal substrates, that act to prevent coding end release or to facilitate coding end recapture.     

Mechanisms of intermolecular homologous recombination in plants as studied with single- and double-stranded DNA molecules.

To elucidate the mechanism for intermolecular homologous recombination in plants we cotransformed Nicotiana tabacum cv Petit Havana SR1 protoplasts with constructs carrying different defective derivatives of the NPTII gene. The resulting kanamycin resistant clones were screened for possible recombination products by PCR, which proved to be a valuable technique for this analysis. Our results show that the double-stranded circular DNA molecules used in this study recombine predominantly via a pathway consistent with the single-strand annealing (SSA) model as proposed for extrachromosomal recombination in mammalian cells. In the remaining cases recombination occurred via a single reciprocal recombination, gene conversion and possibly double reciprocal recombination. Since single-stranded DNA is considered to be an important intermediate in homologous recombination we also established the recombination ability of single-stranded DNA in intermolecular recombination. We found that single-stranded DNA enters in recombination processes more efficiently than the corresponding double-stranded DNA. This was also reflected in the recombination mechanisms that generated the functional NPTII gene. Recombination between a single-stranded DNA and the complementing DNA duplex occurred at similar rates via a single reciprocal recombination and the SSA pathway.     

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker. It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector. The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes. Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination. Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one. The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se. Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one. We propose a model to account for the generation of these recombinants.     

AAV Recombineering with Single Strand Oligonucleotides

Adeno-associated virus (AAV) transduction initiates a signaling cascade that culminates in a transient DNA damage response. During this time, host DNA repair proteins convert the linear single-strand AAV genomes to double-strand circular monomers and concatemers in processes stimulated by the AAV inverted terminal repeats (ITRs). As the orientation of AAV genome concatemerization appears unbiased, the likelihood of concatemerization in a desired orientation is low (less than 1 in 6). Using a novel recombineering method, Oligo-Assisted AAV Genome Recombination (OAGR), this work demonstrates the ability to direct concatemerization specifically to a desired orientation in human cells. This was achieved by a single-strand DNA oligonucleotide (oligo) displaying homology to distinct AAV genomes capable of forming an intermolecular bridge for recombination. This DNA repair process results in concatemers with genomic junctions corresponding to the sequence of oligo homology. Furthermore, OAGR was restricted to single-strand, not duplexed, AAV genomes suggestive of replication-dependent recombination. Consistent with this process, OAGR demonstrated oligo polarity biases in all tested configurations except when a portion of the oligo targeted the ITR. This approach, in addition to being useful for the elucidation of intermolecular homologous recombination, may find eventual relevance for AAV mediated large gene therapy.     

High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication

Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.     

The FLP protein of the 2-micron plasmid of yeast. Inter- and intramolecular reactions

We have studied the mechanism of reaction of the FLP protein of the yeast 2-micron plasmid on linear substrates. The products of the reaction are dependent upon the concentration of FLP protein. At low concentrations of FLP, products resulting from intramolecular recombination between two FLP target sites accumulate. At higher concentrations of FLP, intermolecular recombination results in the accumulation of products which are larger than the starting substrate. At higher concentrations still, FLP-promoted recombination is inhibited. Potassium chloride (0.15 M) inhibits the intermolecular reaction and also prevents the inhibition of FLP-mediated recombination caused by high concentrations of FLP protein. We present a model that explains these findings.     

Site-specific recombination in eukaryotic cells mediated by mutant lambda integrases: implications for synaptic complex formation and the reactivity of episomal DNA segments

Mutant lambda integrases catalyze site-specific DNA recombination in the absence of accessory factors IHF, XIS, and negative DNA supercoiling. Here we investigate the effects that a human cellular environment exerts on these reactions in order to (i) gain further insights into mechanistic aspects of recombination in eukaryotic cells and (ii) to further develop the Int system for biotechnological applications. First, we compared intra- and intermolecular integrative as well as excisive recombination pathways on episomal substrates after co-transfection with recombinase expression vectors. Our results demonstrate that, within 24 hours after transfection, intermolecular recombination by mutant integrase is at least as efficient as intramolecular recombination. Second, a significant intermolecular recombination activity was observed between two copies of a recombination site containing only the 21 bp comprising core-type DNA sequence. This basic activity was stimulated several-fold when arm-type DNA sequences were present in addition to core sites. Therefore, one recombination pathway in human cells involves mutant integrases bound solely at core sites, which is reminiscent of the Flp/FRT and Cre/loxP pathways. The stimulatory effect of arm-type sequences could be explained by an increase in integrase concentration in the vicinity of core sites. We show, in addition, that an N-terminal truncated mutant integrase exhibited only a very weak recombinogenic activity in a eukaryotic background. This result strengthens a functional role for the N-terminal domain in recombination in addition to its arm-type DNA-binding activity. Finally, we demonstrate that low level integrative recombination by wild-type integrase is stimulated when purified integration host factor is co-transfected. This corroborates our previous conclusion that sufficient amounts of eukaryotic protein co-factors, which could functionally replace IHF, are not present in human cells. It also provides a potential means to control site-specific recombination in eukaryotic cells.     

Intermolecular V(D)J recombination is prohibited specifically at the joining step

V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements.     

Linking-number changes in the DNA substrate during Cre-mediated loxP site-specific recombination

We have examined the linking-number changes that occur during phage P1 Cre-mediated recombination in vitro between two loxP sites. Such recombination reactions can be divided into three types: intramolecular inversion, in which recombination occurs between two loxP sites in opposite orientations on the same DNA substrate; intramolecular excision, where recombination occurs between two loxP sites that are in the same orientation on the DNA substrate; and intermolecular recombination, which occurs between two loxP sites on separate DNA molecules. Our results indicate that inversion changes the linking number of the substrate DNA by two topological turns. With a negatively supercoiled substrate, the product is changed by +2 turns. A relaxed substrate yields products that have been changed by either +2 or -2 turns. For intermolecular reactions, the sum of the linking numbers of each of the two starting circles is equal to the linking number of the dimer circle generated by recombination, and no change occurs in linking number. For intramolecular excision reactions, the data are most consistent, with no change in linking number during recombination. These results are discussed in terms of models for alignment and synapsis of the recombining sites and the mechanism of strand exchange.     

In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase

The site-specific integrase of actinophage R4 belongs to the serine recombinase family. During the lysogenization process, it catalyzes site-specific recombination between the phage genome and the chromosome of Streptomyces parvulus 2297. An in vivo assay using Escherichia coli cells revealed that the minimum lengths of the recombination sites attB and attP are 50-bp and 49-bp, respectively, for efficient intramolecular recombination. The in vitro assay using overproduced R4 integrases as a hexahistidine (His(6))-glutathione-S-transferase (GST)-R4 integrase fusion protein, showed that the purified protein preparation retains the site-specific recombination activity which catalyzes the site-specific recombination between attP and attB in the intermolecular reaction. It also revealed that the inverted repeat within attP is essential for efficient in vitro intermolecular recombination. In addition, a gel shift assay showed that His(6)-GST-R4 integrase bound to the 50-bp attB and 49-bp attP specifically. Moreover, based on a detailed comparison analysis of amino acid sequences of serine integrases, we found the DNA binding region that is conserved in the serine recombinase containing the large C-terminal domain. Based on the results presented on this report, attachment sites needed in vitro and in vivo for site-specific recombination by the R4 integrase have been defined more precisely. This knowledge is useful for developing new genetic manipulation tools in the future.     

Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes

The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a beta-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute beta-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.     

Evidence for Retroviral Intramolecular Recombinations

As a consequence of being diploid, retroviruses have a high recombination rate. Naturally occurring retroviruses contain two repeat sequences (R regions) flanking either end of their RNA genomes, and recombination between these two R regions occurs at a high rate. We deduced that recombination may occur between two sequences within the same RNA molecule (intramolecular) as well as between sequences present within two separate RNA molecules (intermolecular). Intramolecular recombination would usually result in a deletion within the progeny provirus. In this report, we demonstrate that intramolecular recombination between two identical sequences occurred within a chimeric RNA vector. In addition, high rates of recombination between two identical sequences within the same RNA molecule resulted mostly from intramolecular recombination.     

Triplex-induced recombination in human cell-free extracts. Dependence on XPA and HsRad51

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 x 10(-)5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51.     

Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase

Cre is a 38-kD protein from bacteriophage P1 that catalyzes site-specific recombination between 34-bp loxP sequences. Our previous work has shown that Cre can perform site-specific excisive recombination not only in prokaryotes, but also in eukaryotes such as yeast and cultured mammalian cells. In this work we show that intermolecular Cre-mediated recombination can specifically direct the integration of a loxP-containing circular DNA into a chromosomal loxP site, both in yeast and in mammalian cells. The resulting integrants are predominantly simple single-copy insertions. Cre-mediated recombination thus provides a simple way to direct single-copy site-specific integration of exogenous DNA into the eukaryotic genome.